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Sample records for affect peptide identification

  1. Peptide identification

    DOEpatents

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  2. Accurate Peptide Fragment Mass Analysis: Multiplexed Peptide Identification and Quantification

    PubMed Central

    Weisbrod, Chad R.; Eng, Jimmy K.; Hoopmann, Michael R.; Baker, Tahmina; Bruce, James E.

    2012-01-01

    FT All Reaction Monitoring (FT-ARM) is a novel approach for the identification and quantification of peptides that relies upon the selectivity of high mass accuracy data and the specificity of peptide fragmentation patterns. An FT-ARM experiment involves continuous, data-independent, high mass accuracy MS/MS acquisition spanning a defined m/z range. Custom software was developed to search peptides against the multiplexed fragmentation spectra by comparing theoretical or empirical fragment ions against every fragmentation spectrum across the entire acquisition. A dot product score is calculated against each spectrum in order to generate a score chromatogram used for both identification and quantification. Chromatographic elution profile characteristics are not used to cluster precursor peptide signals to their respective fragment ions. FT-ARM identifications are demonstrated to be complementary to conventional data-dependent shotgun analysis, especially in cases where the data-dependent method fails due to fragmenting multiple overlapping precursors. The sensitivity, robustness and specificity of FT-ARM quantification are shown to be analogous to selected reaction monitoring-based peptide quantification with the added benefit of minimal assay development. Thus, FT-ARM is demonstrated to be a novel and complementary data acquisition, identification, and quantification method for the large scale analysis of peptides. PMID:22288382

  3. Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

    SciTech Connect

    Alpert, Andrew J.; Petritis, Konstantinos; Kangas, Lars J.; Smith, Richard D.; Mechtler, Karl; Mitulovic, Goran; Mohammed, Shabaz; Heck, Albert J.

    2010-06-15

    Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of harged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptide’s net charge rather than its sequence. These general observations could be of value in confirming a peptide’s identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same compositionbut different sequence.

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

    PubMed

    Shanmugam, Avinash K; Nesvizhskii, Alexey I

    2015-12-01

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

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

    PubMed

    Shanmugam, Avinash K; Nesvizhskii, Alexey I

    2015-12-01

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

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

    PubMed

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

    2013-01-01

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

  7. Bilingualism affects audiovisual phoneme identification.

    PubMed

    Burfin, Sabine; Pascalis, Olivier; Ruiz Tada, Elisa; Costa, Albert; Savariaux, Christophe; Kandel, Sonia

    2014-01-01

    We all go through a process of perceptual narrowing for phoneme identification. As we become experts in the languages we hear in our environment we lose the ability to identify phonemes that do not exist in our native phonological inventory. This research examined how linguistic experience-i.e., the exposure to a double phonological code during childhood-affects the visual processes involved in non-native phoneme identification in audiovisual speech perception. We conducted a phoneme identification experiment with bilingual and monolingual adult participants. It was an ABX task involving a Bengali dental-retroflex contrast that does not exist in any of the participants' languages. The phonemes were presented in audiovisual (AV) and audio-only (A) conditions. The results revealed that in the audio-only condition monolinguals and bilinguals had difficulties in discriminating the retroflex non-native phoneme. They were phonologically "deaf" and assimilated it to the dental phoneme that exists in their native languages. In the audiovisual presentation instead, both groups could overcome the phonological deafness for the retroflex non-native phoneme and identify both Bengali phonemes. However, monolinguals were more accurate and responded quicker than bilinguals. This suggests that bilinguals do not use the same processes as monolinguals to decode visual speech.

  8. 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

  9. Identification of multifunctional peptides from human milk.

    PubMed

    Mandal, Santi M; Bharti, Rashmi; Porto, William F; Gauri, Samiran S; Mandal, Mahitosh; Franco, Octavio L; Ghosh, Ananta K

    2014-06-01

    Pharmaceutical industries have renewed interest in screening multifunctional bioactive peptides as a marketable product in health care applications. In this context, several animal and plant peptides with potential bioactivity have been reported. Milk proteins and peptides have received much attention as a source of health-enhancing components to be incorporated into nutraceuticals and functional foods. By using this source, 24 peptides have been fractionated and purified from human milk using RP-HPLC. Multifunctional roles including antimicrobial, antioxidant and growth stimulating activity have been evaluated in all 24 fractions. Nevertheless, only four fractions show multiple combined activities among them. Using a proteomic approach, two of these four peptides have been identified as lactoferrin derived peptide and kappa casein short chain peptide. Lactoferrin derived peptide (f8) is arginine-rich and kappa casein derived (f12) peptide is proline-rich. Both peptides (f8 and f12) showed antimicrobial activities against both Gram-positive and Gram-negative bacteria. Fraction 8 (f8) exhibits growth stimulating activity in 3T3 cell line and f12 shows higher free radical scavenging activity in comparison to other fractions. Finally, both peptides were in silico evaluated and some insights into their mechanism of action were provided. Thus, results indicate that these identified peptides have multiple biological activities which are valuable for the quick development of the neonate and may be considered as potential biotechnological products for nutraceutical industry.

  10. Synthetic Peptide libraries for T-cell epitope identification.

    PubMed

    Hiemstra, H S; Drijfhout, J W; Koning, F

    2000-01-01

    This chapter describes a methodology for elucidating immunogenic epitopes stimulatory for CD4(+) T-cell clones (Fig. 1). The methodology makes use of synthetic peptide libraries and must be regarded as an alternative to other approaches, such as peptide elution or the application of genetic libraries. The methodology only requires knowledge about the restriction element of the T-cell clone. The restriction element determines which major histocompatibility complex (MHC)-binding anchor motif must be built into the library peptides. A synthetic peptide library is prepared comprising approx 8 million peptides. The synthesis proceeds via a mix-and-split protocol using a solidphase approach on a hybrid resin (1,2). On a hybrid resin, most of the peptide material (84%) is attached via an acid-labile linker whereas the remaining part of the peptide material is acid-stable attached (3). During synthesis, resinbound peptides comprising 14 amino acid residues are produced, with each resin bead containing one unique peptide (4,5). The beads are split into 384 pools, with each pool containing 20,000 beads. From each pool, about 28% of the peptide material is cleaved from every bead. Subsequently, in the first screening round, the 384 pools, each containing 20,000 solubilized peptides, are tested in a proliferation assay with the T-cell clone. Fig. 1. Flow diagram of the complete procedure for the identification of T-cell epitopes using synthetic peptide libraries (1).

  11. Peptidomic Identification of Serum Peptides Diagnosing Preeclampsia

    PubMed Central

    Wu, Shuaibin; Stevenson, David K.; Sheng, Guojun; Butte, Atul J.; Ling, Xuefeng B.

    2013-01-01

    We sought to identify serological markers capable of diagnosing preeclampsia (PE). We performed serum peptide analysis (liquid chromatography mass spectrometry) of 62 unique samples from 31 PE patients and 31 healthy pregnant controls, with two-thirds used as a training set and the other third as a testing set. Differential serum peptide profiling identified 52 significant serum peptides, and a 19-peptide panel collectively discriminating PE in training sets (n = 21 PE, n = 21 control; specificity = 85.7% and sensitivity = 100%) and testing sets (n = 10 PE, n = 10 control; specificity = 80% and sensitivity = 100%). The panel peptides were derived from 6 different protein precursors: 13 from fibrinogen alpha (FGA), 1 from alpha-1-antitrypsin (A1AT), 1 from apolipoprotein L1 (APO-L1), 1 from inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), 2 from kininogen-1 (KNG1), and 1 from thymosin beta-4 (TMSB4). We concluded that serum peptides can accurately discriminate active PE. Measurement of a 19-peptide panel could be performed quickly and in a quantitative mass spectrometric platform available in clinical laboratories. This serum peptide panel quantification could provide clinical utility in predicting PE or differential diagnosis of PE from confounding chronic hypertension. PMID:23840341

  12. Nontargeted identification of peptides and disinfection byproducts in water.

    PubMed

    Tang, Yanan; Xu, Ying; Li, Feng; Jmaiff, Lindsay; Hrudey, Steve E; Li, Xing-Fang

    2016-04-01

    A broad range of organic compounds are known to exist in drinking water sources and serve as precursors of disinfection byproducts (DBPs). Epidemiological findings of an association of increased risk of bladder cancer with the consumption of chlorinated water has resulted in health concerns about DBPs. Peptides are thought to be an important category of DBP precursors in water. However, little is known about the actual presence of peptides and their DBPs in drinking water because of their high sample complexity and low concentrations. To address this challenge and identify peptides and non-chlorinated/chlorinated peptide DBPs from large sets of organic compounds in water, we developed a novel high throughput analysis strategy, which integrated multiple solid phase extraction (SPE), high performance liquid chromatography (HPLC) separation, and non-target identification using precursor ion exclusion (PIE) high resolution mass spectrometry (MS). After MS analysis, structures of candidate compounds, particularly peptides, were obtained by searching against the Human Metabolome Database (HMDB). Using this strategy, we successfully detected 625 peptides (out of 17,205 putative compounds) and 617 peptides (out of 13,297) respectively in source and finished water samples. The source and finished water samples had 501 peptides and amino acids in common. The remaining 116 peptides and amino acids were unique to the finished water. From a subset of 30 putative compounds for which standards were available, 25 were confirmed using HPLC-MS analysis. By analyzing the peptides identified in source and finished water, we successfully confirmed three disinfection reaction pathways that convert peptides into toxic DBPs. PMID:27090718

  13. Identification of Immunodominant Peptides from Gnathostoma binucleatum

    PubMed Central

    Campista-León, Samuel; Delgado-Vargas, Francisco; Landa, Abraham; Willms, Kaethe; López-Moreno, Hector Samuel; Mendoza-Hernández, Guillermo; Ríos-Sicairos, Julian; Bojórquez-Contreras, Ángel Noel; Díaz-Camacho, Sylvia Páz

    2012-01-01

    Gnathostomiasis is now recognized as a zoonosis with a worldwide distribution. In the Americas, it is caused by the third-stage larvae of Gnathostoma binucleatum and in Asia mainly by G. spinigerum. The availability and preparation of specific antigens are among the main obstacles for developing reliable immunodiagnostic tests. In this study, six immunodominant peptides were identified and characterized from G. binucleatum, somatic antigens (AgS: 24, 32, and 40 kDa) and excretory-secretory antigens (AgES: 42, 44, and 56 kDa) by two-dimensional immunoblot analysis. Among those immunodominant peptides, two AgS spots were characterized by mass spectrometric analysis (32 kDa; pI 6.3 and 6.5) and identified as type 1 galectins. In accordance with this finding, a fraction of AgS exhibited affinity to lactose and displayed a 100% specificity and sensitivity for the diagnosis of human gnathostomiasis. PMID:22949520

  14. In silico identification of novel hevein-like peptide precursors.

    PubMed

    Porto, William F; Souza, Valéria A; Nolasco, Diego O; Franco, Octávio L

    2012-11-01

    Lectins are proteins with ability to bind reversibly and non-enzymatically to a specific carbohydrate. They are involved in numerous biological processes and show enormous biotechnological potential. Among plant lectins, the hevein domain is extremely common, being observed in several kinds of lectins. Moreover, this domain is also observed in an important class of antimicrobial peptides named hevein-like peptides. Due to higher cysteine residues conservation, hevein-like peptides could be mined among the sequence databases. By using the pattern CX(4,5)CC[GS]X(2)GXCGX[GST]X(2,3)[FWY]C[GS]X[AGS] novel hevein-like peptide precursors were found from three different plants: Oryza sativa, Vitis vinifera and Selaginella moellendorffii. In addition, an hevein-like peptide precursor from the phytopathogenic fungus Phaeosphaeria nodorum was also identified. The molecular models indicate that they have the same scaffold as others, composed of an antiparallel β-sheet and short helices. Nonetheless, the fungal hevein-like peptide probably has a different disulfide bond pattern. Despite this difference, the complexes between peptide and N,N,N-triacetylglucosamine are stable, according to molecular dynamics simulations. This is the first report of an hevein-like peptide from an organism outside the plant kingdom. The exact role of an hevein-like peptide in the fungal biology must be clarified, while in plants they are clearly involved in plant defense. In summary, data here reported clear shows that an in silico strategy could lead to the identification of novel hevein-like peptides that could be used as biotechnological tools in the fields of health and agribusiness.

  15. Matrix stiffness affects endocytic uptake of MK2-inhibitor peptides.

    PubMed

    Brugnano, Jamie L; Panitch, Alyssa

    2014-01-01

    In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2), enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA) for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10-100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy.

  16. Matrix Stiffness Affects Endocytic Uptake of MK2-Inhibitor Peptides

    PubMed Central

    Brugnano, Jamie L.; Panitch, Alyssa

    2014-01-01

    In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2), enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA) for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10–100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy. PMID:24400117

  17. Identification of Soft Matter Binding Peptide Ligands Using Phage Display.

    PubMed

    Günay, Kemal Arda; Klok, Harm-Anton

    2015-10-21

    Phage display is a powerful tool for the selection of highly affine, short peptide ligands. While originally primarily used for the identification of ligands to proteins, the scope of this technique has significantly expanded over the past two decades. Phage display nowadays is also increasingly applied to identify ligands that selectively bind with high affinity to a broad range of other substrates including natural and biological polymers as well as a variety of low-molecular-weight organic molecules. Such peptides are of interest for various reasons. The ability to selectively and with high affinity bind to the substrate of interest allows the conjugation or immobilization of, e.g., nanoparticles or biomolecules, or generally, facilitates interactions at materials interfaces. On the other hand, presentation of peptide ligands that selectively bind to low-molecular-weight organic materials is of interest for the development of sensor surfaces. The aim of this article is to highlight the opportunities provided by phage display for the identification of peptide ligands that bind to synthetic or natural polymer substrates or to small organic molecules. The article will first provide an overview of the different peptide ligands that have been identified by phage display that bind to these "soft matter" targets. The second part of the article will discuss the different characterization techniques that allow the determination of the affinity of the identified ligands to the respective substrates. PMID:26275106

  18. Pathogen identification using peptide nanotube biosensors and impedance AFM

    NASA Astrophysics Data System (ADS)

    Maccuspie, Robert I.

    Pathogen identification at highly sensitive levels is crucial to meet urgent needs in fighting the spread of disease or detecting bioterrorism events. Toward that end, a new method for biosensing utilizing fluorescent antibody nanotubes is proposed. Fundamental studies on the self-assembly of these peptide nanotubes are performed, as are applications of aligning these nanotubes on surfaces. As biosensors, these nanotubes incorporate recognition units with antibodies at their ends and fluorescent signaling units at their sidewalls. When viral pathogens were mixed with these antibody nanotubes in solution, the nanotubes rapidly aggregated around the viruses. The size of the aggregates increased as the concentration of viruses increased, as detected by flow cytometry on the order of attomolar concentrations by changes in fluorescence and light scattering intensities. This enabled determination of the concentrations of viruses at trace levels (102 to 106 pfu/mL) within 30 minutes from the receipt of samples to the final quantitative data analysis, as demonstrated on Adenovirus, Herpes Simplex Virus, Influenza, and Vaccinia virus. As another separate approach, impedance AFM is used to study the electrical properties of individual viruses and nanoparticles used as model systems. The design, development, and implementation of the impedance AFM for an Asylum Research platform is described, as well as its application towards studying the impedance of individual nanoparticles as a model system for understanding the fundamental science of how the life cycle of a virus affects its electrical properties. In combination, these approaches fill a pressing need to quantify viruses both rapidly and sensitively.

  19. 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.

  20. Data on the peptide mapping and MS identification for phosphorylated peptide.

    PubMed

    Wang, Hui; Tu, Zong-Cai; Liu, Guang-Xian; Zhang, Lu; Chen, Yuan

    2016-09-01

    This article contains peptides mapping, mass spectrometry and processed data related to the research "Identification and quantification of the phosphorylated ovalbumin by high resolution mass spectrometry under dry-heating treatment" [1]. Fourier transform ion cyclotron mass spectrometry (FTICR MS) was used to investigate the specific phosphorylation sites and the degree of phosphorylation (DSP) at each site. Specifically, phosphorylated peptides were monitored through mass shift on the FTICR MS spectrum. DSP was evaluated through the relative abundance levels of the FTICR MS spectrometry. From these data, the calculation method of DSP was exemplified. PMID:27274527

  1. Faster SEQUEST Searching for Peptide Identification from Tandem Mass Spectra

    PubMed Central

    Diament, Benjamin; Noble, William Stafford

    2011-01-01

    Computational analysis of mass spectra remains the bottleneck in many proteomics experiments. SEQUEST was one of the earliest software packages to identify peptides from mass spectra by searching a database of known peptides. Though still popular, SEQUEST performs slowly. Crux and TurboSEQUEST have successfully sped up SEQUEST by adding a precomputed index to the search, but the demand for ever-faster peptide identification software continues to grow. Tide, introduced here, is a software program that implements the SEQUEST algorithm for peptide identification and that achieves a dramatic speedup over Crux and SEQUEST. The optimization strategies detailed here employ a combination of algorithmic and software engineering techniques to achieve speeds up to 170 times faster than a recent version of SEQUEST that uses indexing. For example, on a single Xeon CPU, Tide searches 10,000 spectra against a tryptic database of 27,499 C. elegans proteins at a rate of 1,550 spectra per second, which compares favorably with a rate of 8.8 spectra per second for a recent version of SEQUEST with index running on the same hardware. PMID:21761931

  2. Amyloid Beta Peptides Differentially Affect Hippocampal Theta Rhythms In Vitro

    PubMed Central

    Gutiérrez-Lerma, Armando I.; Ordaz, Benito; Peña-Ortega, Fernando

    2013-01-01

    Soluble amyloid beta peptide (Aβ) is responsible for the early cognitive dysfunction observed in Alzheimer's disease. Both cholinergically and glutamatergically induced hippocampal theta rhythms are related to learning and memory, spatial navigation, and spatial memory. However, these two types of theta rhythms are not identical; they are associated with different behaviors and can be differentially modulated by diverse experimental conditions. Therefore, in this study, we aimed to investigate whether or not application of soluble Aβ alters the two types of theta frequency oscillatory network activity generated in rat hippocampal slices by application of the cholinergic and glutamatergic agonists carbachol or DHPG, respectively. Due to previous evidence that oscillatory activity can be differentially affected by different Aβ peptides, we also compared Aβ25−35 and Aβ1−42 for their effects on theta rhythms in vitro at similar concentrations (0.5 to 1.0 μM). We found that Aβ25−35 reduces, with less potency than Aβ1−42, carbachol-induced population theta oscillatory activity. In contrast, DHPG-induced oscillatory activity was not affected by a high concentration of Aβ25−35 but was reduced by Aβ1−42. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of exerting a generalized inhibitory effect on neuronal network function. PMID:23878547

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

    DOE PAGES

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

    2013-03-07

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

  4. 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.

  5. Factors Affecting Peptide Interactions with Surface-Bound Microgels.

    PubMed

    Nyström, Lina; Nordström, Randi; Bramhill, Jane; Saunders, Brian R; Álvarez-Asencio, Rubén; Rutland, Mark W; Malmsten, Martin

    2016-02-01

    Effects of electrostatics and peptide size on peptide interactions with surface-bound microgels were investigated with ellipsometry, confocal microscopy, and atomic force microscopy (AFM). Results show that binding of cationic poly-L-lysine (pLys) to anionic, covalently immobilized, poly(ethyl acrylate-co-methacrylic acid) microgels increased with increasing peptide net charge and microgel charge density. Furthermore, peptide release was facilitated by decreasing either microgel or peptide charge density. Analogously, increasing ionic strength facilitated peptide release for short peptides. As a result of peptide binding, the surface-bound microgels displayed pronounced deswelling and increased mechanical rigidity, the latter quantified by quantitative nanomechanical mapping. While short pLys was found to penetrate the entire microgel network and to result in almost complete charge neutralization, larger peptides were partially excluded from the microgel network, forming an outer peptide layer on the microgels. As a result of this difference, microgel flattening was more influenced by the lower Mw peptide than the higher. Peptide-induced deswelling was found to be lower for higher Mw pLys, the latter effect not observed for the corresponding microgels in the dispersed state. While the effects of electrostatics on peptide loading and release were similar to those observed for dispersed microgels, there were thus considerable effects of the underlying surface on peptide-induced microgel deswelling, which need to be considered in the design of surface-bound microgels as carriers of peptide loads, for example, in drug delivery or in functionalized biomaterials. PMID:26750986

  6. Enhanced identification of peptides lacking basic residues by LC-ESI-MS/MS analysis of singly charged peptides.

    PubMed

    Biniossek, Martin L; Schilling, Oliver

    2012-05-01

    Peptide sequences lacking basic residues (arginine, lysine, or histidine, referred to as "base-less") are of particular importance in proteomic experiments targeting protein C-termini or employing nontryptic proteases such as GluC or chymotrypsin. We demonstrate enhanced identification of base-less peptides by focused analysis of singly charged precursors in liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Singly charged precursors are often excluded from fragmentation and sequence analysis in LC-MS/MS. We generated different pools of base-less and base-containing peptides by tryptic and nontryptic digestion of bacterial proteomes. Focused LC-MS/MS analysis of singly charged precursor ions yielded predominantly base-less peptide identifications. Similar numbers of base-less peptides were identified by LC-MS/M Sanalysis targeting multiply charged precursors. There was little redundancy between the base-less sequences derived by both MS/MS schemes. In the present experimental outcome, additional LC-MS/MS analysis of singly charged precursors substantially increased the identification rate of base-less sequences derived from multiply charged precursors. In conclusion, LC-MS/MS based identification of base-less peptides is substantially enhanced by additional focused analysis of singly charged precursors.

  7. Application of Peptide LC Retention Time Information in a Discriminant Function for Peptide Identification by Tandem Mass Spectrometry

    SciTech Connect

    Strittmatter, Eric F.; Kangas, Lars J.; Petritis, Konstantinos; Mottaz, Heather M.; Anderson, Gordon A.; Shen, Yufeng; Jacobs, Jon M.; Camp, David G.; Smith, Richard D.

    2004-07-09

    We describe the application of a peptide retention time reversed phase liquid chromatography (RPLC) prediction model previously reported (Petritis et al. Anal. Chem. 99, 2002, 11049) for improved peptide identification. The model uses peptide sequence information to generate a theoretical (predicted) elution time that can be compared with the observed elution time. Using data from a set of known proteins, the retention time parameter was incorporated into a discriminant function for use with tandem mass spectrometry (MS/MS) data analyzed with the peptide/protein identification program SEQUEST. For singly charged ions, the number of identifications increased by 12% when the elution time metric is included compared to when mass spectral data is the sole source of information in the context of a Drosophila melanogaster database. A 3-4% improvement was obtained for doubly and triply charged ions for the same biological system. Application to the larger Rattus norvegicus (rat) and human proteome databases resulted in an 8-9% overall increase in the number of identifications, when both the discriminant function and elution time are used. The effect of adding “runner-up” hits (peptide matches that are not the highest scoring for a spectra) from SEQUEST is also explored, and we find that the number of confident identifications is further increased when these hits are also considered. Finally, application of the discriminant functions derived in this work with ~2.2 million spectra from 330 LC-MS/MS analyses of peptides from human plasma protein resulted in a 19% increase in confident peptide identifications (9551 vs 8049) using elution time information. Further improvements from the use of elution time information can be expected as both the experimental control of elution time reproducibility and the predictive capability are improved.

  8. The importance of peptide detectability for protein identification, quantification, and experiment design in MS/MS proteomics

    PubMed Central

    Li, Yong Fuga; Arnold, Randy J.; Tang, Haixu

    2010-01-01

    Peptide detectability is defined as the probability that a peptide is identified in an LC-MS/MS experiment and has been useful in providing solutions to protein inference and label-free quantification. Previously, predictors for peptide detectability trained on standard or complex samples were proposed. Although the models trained on complex samples may benefit from the large training data sets, it is unclear to what extent they are affected by the unequal abundances of identified proteins. To address this challenge and improve detectability prediction, we present a new algorithm for the iterative learning of peptide detectability from complex mixtures. We provide evidence that the new method approximates detectability with useful accuracy and, based on its design, can be used to interpret the outcome of other learning strategies. We studied the properties of peptides from the bacterium Deinococcus radiodurans and found that at standard quantities, its tryptic peptides can be roughly classified as either detectable or undetectable, with a relatively small fraction having medium detectability. We extend the concept of detectability from peptides to proteins and apply the model to predict the behavior of a replicate LC-MS/MS experiment from a single analysis. Finally, our study summarizes a theoretical framework for peptide/protein identification and label-free quantification. PMID:21067214

  9. Current algorithmic solutions for peptide-based proteomics data generation and identification.

    PubMed

    Hoopmann, Michael R; Moritz, Robert L

    2013-02-01

    Peptide-based proteomic data sets are ever increasing in size and complexity. These data sets provide computational challenges when attempting to quickly analyze spectra and obtain correct protein identifications. Database search and de novo algorithms must consider high-resolution MS/MS spectra and alternative fragmentation methods. Protein inference is a tricky problem when analyzing large data sets of degenerate peptide identifications. Combining multiple algorithms for improved peptide identification puts significant strain on computational systems when investigating large data sets. This review highlights some of the recent developments in peptide and protein identification algorithms for analyzing shotgun mass spectrometry data when encountering the aforementioned hurdles. Also explored are the roles that analytical pipelines, public spectral libraries, and cloud computing play in the evolution of peptide-based proteomics.

  10. Identification of the agr Peptide of Listeria monocytogenes.

    PubMed

    Zetzmann, Marion; Sánchez-Kopper, Andrés; Waidmann, Mark S; Blombach, Bastian; Riedel, Christian U

    2016-01-01

    Listeria monocytogenes (Lm) is an important food-borne human pathogen that is able to strive under a wide range of environmental conditions. Its accessory gene regulator (agr) system was shown to impact on biofilm formation and virulence and has been proposed as one of the regulatory mechanisms involved in adaptation to these changing environments. The Lm agr operon is homologous to the Staphylococcus aureus system, which includes an agrD-encoded autoinducing peptide that stimulates expression of the agr genes via the AgrCA two-component system and is required for regulation of target genes. The aim of the present study was to identify the native autoinducing peptide (AIP) of Lm using a luciferase reporter system in wildtype and agrD deficient strains, rational design of synthetic peptides and mass spectrometry. Upon deletion of agrD, luciferase reporter activity driven by the PII promoter of the agr operon was completely abolished and this defect was restored by co-cultivation of the agrD-negative reporter strain with a producer strain. Based on the sequence and structures of known AIPs of other organisms, a set of potential Lm AIPs was designed and tested for PII-activation. This led to the identification of a cyclic pentapeptide that was able to induce PII-driven luciferase reporter activity and restore defective invasion of the agrD deletion mutant into Caco-2 cells. Analysis of supernatants of a recombinant Escherichia coli strain expressing AgrBD identified a peptide identical in mass and charge to the cyclic pentapeptide. The Lm agr system is specific for this pentapeptide since the AIP of Lactobacillus plantarum, which also is a pentapeptide yet with different amino acid sequence, did not induce PII activity. In summary, the presented results provide further evidence for the hypothesis that the agrD gene of Lm encodes a secreted AIP responsible for autoregulation of the agr system of Lm. Additionally, the structure of the native Lm AIP was identified. PMID

  11. Identification of the agr Peptide of Listeria monocytogenes

    PubMed Central

    Zetzmann, Marion; Sánchez-Kopper, Andrés; Waidmann, Mark S.; Blombach, Bastian; Riedel, Christian U.

    2016-01-01

    Listeria monocytogenes (Lm) is an important food-borne human pathogen that is able to strive under a wide range of environmental conditions. Its accessory gene regulator (agr) system was shown to impact on biofilm formation and virulence and has been proposed as one of the regulatory mechanisms involved in adaptation to these changing environments. The Lm agr operon is homologous to the Staphylococcus aureus system, which includes an agrD-encoded autoinducing peptide that stimulates expression of the agr genes via the AgrCA two-component system and is required for regulation of target genes. The aim of the present study was to identify the native autoinducing peptide (AIP) of Lm using a luciferase reporter system in wildtype and agrD deficient strains, rational design of synthetic peptides and mass spectrometry. Upon deletion of agrD, luciferase reporter activity driven by the PII promoter of the agr operon was completely abolished and this defect was restored by co-cultivation of the agrD-negative reporter strain with a producer strain. Based on the sequence and structures of known AIPs of other organisms, a set of potential Lm AIPs was designed and tested for PII-activation. This led to the identification of a cyclic pentapeptide that was able to induce PII-driven luciferase reporter activity and restore defective invasion of the agrD deletion mutant into Caco-2 cells. Analysis of supernatants of a recombinant Escherichia coli strain expressing AgrBD identified a peptide identical in mass and charge to the cyclic pentapeptide. The Lm agr system is specific for this pentapeptide since the AIP of Lactobacillus plantarum, which also is a pentapeptide yet with different amino acid sequence, did not induce PII activity. In summary, the presented results provide further evidence for the hypothesis that the agrD gene of Lm encodes a secreted AIP responsible for autoregulation of the agr system of Lm. Additionally, the structure of the native Lm AIP was identified. PMID

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

    PubMed Central

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

    2009-01-01

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

  13. Constrained De-Novo Peptide Identification via Multi-objective Optimization

    SciTech Connect

    Malard, Joel M.; Heredia-Langner, Alejandro; Baxter, Douglas J.; Jarman, Kristin H.; Cannon, William R.

    2004-04-27

    Automatic de novo peptide identification from collision-induced dissociation tandem mass spectrometry data is made difficult by large plateaus in the fitness landscapes of scoring functions and the fuzzy nature of the constraints that is due to noise in the data. A framework is presented for combining different peptide identification methods within a parallel genetic algorithm. The distinctive feature of our approach, based on Pareto ranking, is that it can accommodate constraints and possibly conflicting scoring functions. We have also shown how population structure can significantly improve the wall clock time of a parallel peptide identification genetic algorithm while at the same time maintaining some exchange of information across local populations.

  14. 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

  15. Identification of peptide-specific TCR genes by in vitro peptide stimulation and CDR3 length polymorphism analysis.

    PubMed

    Shao, Hongwei; Lin, Yanmei; Wang, Teng; Ou, Yusheng; Shen, Han; Tao, Changli; Wu, Fenglin; Zhang, Wenfeng; Bo, Huaben; Wang, Hui; Huang, Shulin

    2015-07-10

    Identification of TCR genes specific for tumor-associated antigens (TAAs) is necessary for TCR gene modification of T cells, which is applied in anti-tumor adoptive T cell therapy (ACT). The usual identification methods are based on isolating single peptide-responding T cells and cloning the TCR gene by in vitro expansion or by single-cell RT-PCR. However, the long and exacting in vitro culture period and demanding operational requirements restrict the application of these methods. Immunoscope is an effective tool that profiles a repertoire of TCRs and identifies significantly expanded clones through CDR3 length analysis. In this study, a survivin-derived mutant peptide optimized for HLA-A2 binding was selected to load DCs and activate T cells. The monoclonal expansion of TCRA and TCRB genes was separately identified by Immunoscope analysis and following sequence identification, the properly paired TCR genes were transferred into T cells. Peptide recognition and cytotoxicity assays indicated that TCR-modified PBMCs could respond to both the mutant and wild type peptides and lyse target cells. These results show that combining Immunoscope with in vitro peptide stimulation provides an alternative and superior method for identifying specific TCR genes, which represents a significant advance for the application of TCR gene-modified T cells. PMID:25890221

  16. Functional region identification in proteins by accumulative-quantitative peptide mapping using RP-HPLC-MS.

    PubMed

    Kuipers, Bas J H; Bakx, E J; Gruppen, Harry

    2007-11-14

    A new method was developed to identify regions in proteins from which peptides are derived with specific functional properties. This method is applicable for systems in which peptides of a hydrolyzed protein possess specific functional properties, but are too large to be sequenced directly and/or the peptide mixture is too complex to purify and characterize each peptide individually. In the present work, aggregating peptides obtained by proteolytic hydrolysis of soy glycinin were used as a case study. The aggregating peptides are isolated and subsequently further degraded with trypsin to result in peptides with a mass <5000 Da to enable sequence identification using RP-HPLC-MS in combination with MS/MS. Prior to RP-HPLC the peptides are fractionated using anion and cation exchange chromatography. The fractions obtained are analyzed with RP-HPLC-MS. The peptides, with identified sequences, were quantified using the peak areas of the RP-HPLC chromatograms measured at 214 nm. Next, the peak areas were corrected for the molar extinction coefficient of the individual peptides, followed by accumulative-quantitative peptide mapping. The results show that in complex systems, based on the method described, the regions in the parental protein from which the functional peptides originate can be properly identified.

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

    PubMed

    Alves, Gelio; Yu, Yi-Kuo

    2013-06-01

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

  18. Peptide identification by database search of mixture tandem mass spectra.

    PubMed

    Wang, Jian; Bourne, Philip E; Bandeira, Nuno

    2011-12-01

    In high-throughput proteomics the development of computational methods and novel experimental strategies often rely on each other. In certain areas, mass spectrometry methods for data acquisition are ahead of computational methods to interpret the resulting tandem mass spectra. Particularly, although there are numerous situations in which a mixture tandem mass spectrum can contain fragment ions from two or more peptides, nearly all database search tools still make the assumption that each tandem mass spectrum comes from one peptide. Common examples include mixture spectra from co-eluting peptides in complex samples, spectra generated from data-independent acquisition methods, and spectra from peptides with complex post-translational modifications. We propose a new database search tool (MixDB) that is able to identify mixture tandem mass spectra from more than one peptide. We show that peptides can be reliably identified with up to 95% accuracy from mixture spectra while considering only a 0.01% of all possible peptide pairs (four orders of magnitude speedup). Comparison with current database search methods indicates that our approach has better or comparable sensitivity and precision at identifying single-peptide spectra while simultaneously being able to identify 38% more peptides from mixture spectra at significantly higher precision.

  19. Identification of peptides that selectively bind to myoglobin by biopanning of phage displayed-peptide library.

    PubMed

    Padmanaban, Guruprasath; Park, Hyekyung; Choi, Ji Suk; Cho, Yong-Woo; Kang, Woong Chol; Moon, Chan-Il; Kim, In-San; Lee, Byung-Heon

    2014-10-10

    Biopanning of phage displayed-peptide library was performed against myoglobin, a marker for the early assessment of acute myocardial infarction (AMI), to identify peptides that selectively bind to myoglobin. Using myoglobin-conjugated magnetic beads, phages that bound to myoglobin were collected and amplified for the next round of screening. A 148-fold enrichment of phage titer was observed after five rounds of screening relative to the first round. After phage binding ELISA, three phage clones were selected (3R1, 3R7 and 3R10) and the inserted peptides were chemically synthesized. The analysis of binding affinity showed that the 3R7 (CPSTLGASC) peptide had higher binding affinity (Kd=57 nM) than did the 3R1 (CNLSSSWIC) and 3R10 (CVPRLSAPC) peptide (Kd=125 nM and 293 nM, respectively). Cross binding activity to other proteins, such as bovine serum albumin, troponin I, and creatine kinase-MB, was minimal. In a peptide-antibody sandwich ELISA, the selected peptides efficiently captured myoglobin. Moreover, the concentrations of myoglobin in serum samples measured by a peptide-peptide sandwich assay were comparable to those measured by a commercial antibody-based kit. These results indicate that the identified peptides can be used for the detection of myoglobin and may be a cost effective alternative to antibodies.

  20. Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification.

    PubMed

    Guixer, B; Arroyo, X; Belda, I; Sabidó, E; Teixidó, M; Giralt, E

    2016-09-01

    The blood-brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma-targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high-throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d-amino acids, this approach screens only protease-resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state-of-the-art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix-and-split technique to generate a library based on the following: Ac-d-Arg-XXXXX-NH2 , where X were: d-Ala (a), d-Arg (r), d-Ile (i), d-Glu (e), d-Ser (s), d-Trp (w) or d-Pro (p). The assays used comprised the in vitro cell-based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two-step mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease-resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  1. Identification of peptides that bind to irradiated pancreatic tumor cells

    SciTech Connect

    Huang Canhui; Liu, Xiang Y.; Rehemtulla, Alnawaz; Lawrence, Theodore S. . E-mail: tsl@med.umich.edu

    2005-08-01

    Purpose: Peptides targeting tumor vascular cells or tumor cells themselves have the potential to be used as vectors for delivering either DNA in gene therapy or antitumor agents in chemotherapy. We wished to determine if peptides identified by phage display could be used to target irradiated pancreatic cancer cells. Methods and Materials: Irradiated Capan-2 cells were incubated with 5 x 10{sup 12} plaque-forming units of a phage display library. Internalized phage were recovered and absorbed against unirradiated cells. After five such cycles of enrichment, the recovered phage were subjected to DNA sequencing analysis and synthetic peptides made. The binding of both phage and synthetic peptides was evaluated by fluorescence staining and flow cytometry in vitro and in vivo. Results: We identified one 12-mer peptide (PA1) that binds to irradiated Capan-2 pancreatic adenocarcinoma cells but not to unirradiated cells. The binding of peptide was significant after 48 h incubation with cells. In vivo experiments with Capan-2 xenografts in nude mice demonstrated that these small peptides are able to penetrate tumor tissue after intravenous injections and bind specifically to irradiated tumor cells. Conclusion: These data suggest that peptides can be identified that target tumors with radiation-induced cell markers and may be clinically useful.

  2. Correlation of Multiple Peptide Mass Spectra for Phosphoprotein Identification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    When collision induced dissociation is used to fragment phosphorylated peptides during tandem mass spectrometry (MS2), an ion exhibiting the neutral loss of phosphoric acid can be the major product. The neutral loss ion can then be fragmented during MS3 for additional resolution of the peptide sequ...

  3. Identification of Chondrocyte-Binding Peptides by Phage Display

    PubMed Central

    Cheung, Crystal S.F.; Lui, Julian C.; Baron, Jeffrey

    2016-01-01

    As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders. PMID:23440926

  4. Identification of D-peptide ligands through mirror-image phage display.

    PubMed

    Schumacher, T N; Mayr, L M; Minor, D L; Milhollen, M A; Burgess, M W; Kim, P S

    1996-03-29

    Genetically encoded libraries of peptides and oligonucleotides are well suited for the identification of ligands for many macromolecules. A major drawback of these techniques is that the resultant ligands are subject to degradation by naturally occurring enzymes. Here, a method is described that uses a biologically encoded library for the identification of D-peptide ligands, which should be resistant to proteolytic degradation. In this approach, a protein is synthesized in the D-amino acid configuration and used to select peptides from a phage display library expressing random L-amino acid peptides. For reasons of symmetry, the mirror images of these phage-displayed peptides interact with the target protein of the natural handedness. The value of this approach was demonstrated by the identification of a cyclic D-peptide that interacts with the Src homology 3 domain of c- SRC. Nuclear magnetic resonance studies indicate that the binding site for this D-peptide partially overlaps the site for the physiological ligands of this domain.

  5. 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

  6. A Novel Algorithm for Validating Peptide Identification from a Shotgun Proteomics Search Engine

    PubMed Central

    Jian, Ling; Niu, Xinnan; Xia, Zhonghang; Samir, Parimal; Sumanasekera, Chiranthani; Zheng, Mu; Jennings, Jennifer L.; Hoek, Kristen L.; Allos, Tara; Howard., Leigh M.; Edwards, Kathryn M.; Weil, P. Anthony; Link, Andrew J.

    2013-01-01

    Liquid chromatography coupled with tandem mass spectrometry has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC/MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm based on the resolution and mass accuracy of the mass spectrometer employed in the LC/MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines. PMID:23402659

  7. A novel algorithm for validating peptide identification from a shotgun proteomics search engine.

    PubMed

    Jian, Ling; Niu, Xinnan; Xia, Zhonghang; Samir, Parimal; Sumanasekera, Chiranthani; Mu, Zheng; Jennings, Jennifer L; Hoek, Kristen L; Allos, Tara; Howard, Leigh M; Edwards, Kathryn M; Weil, P Anthony; Link, Andrew J

    2013-03-01

    Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC-MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three-step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm on the basis of the resolution and mass accuracy of the mass spectrometer employed in the LC-MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines.

  8. A novel algorithm for validating peptide identification from a shotgun proteomics search engine.

    PubMed

    Jian, Ling; Niu, Xinnan; Xia, Zhonghang; Samir, Parimal; Sumanasekera, Chiranthani; Mu, Zheng; Jennings, Jennifer L; Hoek, Kristen L; Allos, Tara; Howard, Leigh M; Edwards, Kathryn M; Weil, P Anthony; Link, Andrew J

    2013-03-01

    Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC-MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three-step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm on the basis of the resolution and mass accuracy of the mass spectrometer employed in the LC-MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines. PMID:23402659

  9. PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS.

    PubMed

    Stokes, Matthew P; Farnsworth, Charles L; Moritz, Albrecht; Silva, Jeffrey C; Jia, Xiaoying; Lee, Kimberly A; Guo, Ailan; Polakiewicz, Roberto D; Comb, Michael J

    2012-05-01

    Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in

  10. Enrichment by organomercurial agarose and identification of cys-containing peptides from yeast cell lysates.

    PubMed

    Raftery, Mark J

    2008-05-01

    Dynamic range and the presence of highly abundant proteins limit the number of proteins that may be identified within a complex mixture. Cysteine (Cys) has unique chemical reactivity that may be exploited for chemical tagging/capture with biotin/avidin reagents or affinity chromatography allowing specific isolation and subsequent identification of peptide sequences by mass spectrometry. Organomercurial agarose (Hg-beads) specifically captures Cys-containing peptides and proteins from cell lysates. Tryptic peptides from yeast lysates containing Cys were captured and eluted from Hg-beads after incubation with TCEP and trypsin. From two 1 h nano 1-D LC DDA/MS of the eluate >700 proteins were identified with an estimated false positive rate of approximately 1%. Few peptides were identified with high confidence without Cys within their sequence after capture, and extensive washing, indicating little nonspecific binding. The number of fragmentation spectra was increased using automated 2-D nano-LC/MS and allowed identification of 1496 proteins with an estimated false positive rate of 1.1%. Approximately 4% of the proteins identified were from peptides that did not contain Cys, and these were biased toward higher abundance proteins. Comparison of the 1496 proteins to those reported previously showed that >25% were from yeast proteins not previously observed. Most proteins were identified from a single peptide, and sequence coverage was sacrificed by focusing only on identifying Cys-containing peptides, but large numbers of proteins were rapidly identified by eliminating many of the peptides from the higher abundance proteins.

  11. PepArML: A Meta-Search Peptide Identification Platform

    PubMed Central

    Edwards, Nathan J.

    2014-01-01

    The PepArML meta-search peptide identification platform provides a unified search interface to seven search engines; a robust cluster, grid, and cloud computing scheduler for large-scale searches; and an unsupervised, model-free, machine-learning-based result combiner, which selects the best peptide identification for each spectrum, estimates false-discovery rates, and outputs pepXML format identifications. The meta-search platform supports Mascot; Tandem with native, k-score, and s-score scoring; OMSSA; MyriMatch; and InsPecT with MS-GF spectral probability scores — reformatting spectral data and constructing search configurations for each search engine on the fly. The combiner selects the best peptide identification for each spectrum based on search engine results and features that model enzymatic digestion, retention time, precursor isotope clusters, mass accuracy, and proteotypic peptide properties, requiring no prior knowledge of feature utility or weighting. The PepArML meta-search peptide identification platform often identifies 2–3 times more spectra than individual search engines at 10% FDR. PMID:25663956

  12. PepArML: A Meta-Search Peptide Identification Platform for Tandem Mass Spectra.

    PubMed

    Edwards, Nathan J

    2013-12-01

    The PepArML meta-search peptide identification platform for tandem mass spectra provides a unified search interface to seven search engines; a robust cluster, grid, and cloud computing scheduler for large-scale searches; and an unsupervised, model-free, machine-learning-based result combiner, which selects the best peptide identification for each spectrum, estimates false-discovery rates, and outputs pepXML format identifications. The meta-search platform supports Mascot; Tandem with native, k-score and s-score scoring; OMSSA; MyriMatch; and InsPecT with MS-GF spectral probability scores—reformatting spectral data and constructing search configurations for each search engine on the fly. The combiner selects the best peptide identification for each spectrum based on search engine results and features that model enzymatic digestion, retention time, precursor isotope clusters, mass accuracy, and proteotypic peptide properties, requiring no prior knowledge of feature utility or weighting. The PepArML meta-search peptide identification platform often identifies two to three times more spectra than individual search engines at 10% FDR.

  13. Identification of peptides in functional Scamorza ovine milk cheese.

    PubMed

    Albenzio, M; Santillo, A; Marino, R; Della Malva, A; Caroprese, M; Sevi, A

    2015-12-01

    Ovine bulk milk was used to produce Scamorza cheese with probiotics: either a mix of Bifidobacterium longum and Bifidobacterium lactis or Lactobacillus acidophilus as the probiotic strains. Peptides obtained from reverse phase-HPLC water-soluble extract of Scamorza cheeses were analyzed using a quadrupole time-of-flight liquid chromatography-mass spectrometry system. Identified fragments were derived from casein hydrolysis or probiotic bacterial enzymes; some of the fragments showed encrypted peptide sequences that shared structural homology with previously described bioactive peptides in ovine milk and dairy products. Bifidobacterium longum and B. lactis showed greater proteolytic potential both in terms of level of pH 4.6 water-soluble nitrogen extract and ability to generate peptides with potential biofunctionality. Fragments deriving from microbial enzymes may be regarded as tracing fragments useful for monitoring probiotic activity in functional Scamorza cheese. PMID:26409967

  14. 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.

  15. Identification of bioactive peptide from Oreochromis niloticus skin gelatin.

    PubMed

    Choonpicharn, Sadabpong; Tateing, Suriya; Jaturasitha, Sanchai; Rakariyatham, Nuansri; Suree, Nuttee; Niamsup, Hataichanoke

    2016-02-01

    Fish skin, one type of wastes generated from Nile tilapia processing, is still a good source of collagen and gelatin. Bioactive peptides can be obtained from Nile tilapia skin gelatin by trypsin digestion. Trypsin hydrolysate was subsequently purified by gel filtration chromatography. Trypsin A fraction showed the greatest reducing power (5.138 ± 1.060 μM trolox/mg peptide) among all hydrolysate fractions, while trypsin B fraction from gel filtration column was found to exhibit the best radical scavenging and angiotensin-I-converting enzyme (ACE) inhibitory activities 8.16 ± 2.18 μg trolox/mg peptide and 59.32 ± 9.97 % inhibition, respectively. The most active fraction was subjected to MALDI-TOF/TOF MS/MS. After annotation by Mascot sequence matching software (Matrix Science) with Ludwig NR Database, two peptide sequences were identified; GPEGPAGAR (MW 810.87 Da) and GETGPAGPAGAAGPAGPR (MW 1490.61 Da). The docking analysis suggested that the shape of the shorter peptide may be slightly more proper, to fit into the binding cleft of the ACE. However, the binding affinities calculated from the docking showed no significant difference between the two peptides. In good agreement with the in silico data, results from the in vitro ACE inhibitory activity with synthetic peptides also showed no significant difference. Both peptides are thus interesting novel candidates suitable for further development as ACE inhibitory and antioxidant agents from the natural source. PMID:27162402

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

    SciTech Connect

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

    2011-07-15

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

  17. Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS.

    PubMed

    Pashkova, Anna; Chen, Hsuan-Shen; Rejtar, Tomas; Zang, Xin; Giese, Roger; Andreev, Victor; Moskovets, Eugene; Karger, Barry L

    2005-04-01

    The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.

  18. Identification of candidate antimicrobial peptides derived from abalone hemocyanin.

    PubMed

    Zhuang, Jun; Coates, Christopher J; Zhu, Hongtao; Zhu, Ping; Wu, Zujian; Xie, Lianhui

    2015-03-01

    Hemocyanins present in invertebrate hemolymph are multifunctional proteins, responsible for oxygen transport and contributing to innate immunity through phenoloxidase-like activity. In arthropods, hemocyanin has been identified as a source of broad-spectrum antimicrobial peptides during infection. Conversely, no hemocyanin-derived antimicrobial peptides have been reported for molluscs. The present study describes a putative antimicrobial region, termed haliotisin, located within the linking sequence between the α-helical domain and β-sheet domain of abalone (Haliotis tuberculata) hemocyanin functional unit E. A series of synthetic peptides based on overlapping fragments of the haliotisin region were tested for their bactericidal potential. Incubating Gram-positive and Gram-negative bacteria in the presence of certain haliotisin peptides, notably peptides 3-4-5 (DTFDYKKFGYRYDSLELEGRSISRIDELIQQRQEKDRTFAGFLLKGFGTSAS) led to reductions in microbial growth. Furthermore, transmission electron micrographs of haliotisin-treated bacteria revealed damages to the microbial cell wall. Data discussed here provides the first evidence to suggest that molluscan hemocyanin may act as a source of anti-infective peptides. PMID:25445903

  19. Combinatorial approach for large-scale identification of linked peptides from tandem mass spectrometry spectra.

    PubMed

    Wang, Jian; Anania, Veronica G; Knott, Jeff; Rush, John; Lill, Jennie R; Bourne, Philip E; Bandeira, Nuno

    2014-04-01

    The combination of chemical cross-linking and mass spectrometry has recently been shown to constitute a powerful tool for studying protein-protein interactions and elucidating the structure of large protein complexes. However, computational methods for interpreting the complex MS/MS spectra from linked peptides are still in their infancy, making the high-throughput application of this approach largely impractical. Because of the lack of large annotated datasets, most current approaches do not capture the specific fragmentation patterns of linked peptides and therefore are not optimal for the identification of cross-linked peptides. Here we propose a generic approach to address this problem and demonstrate it using disulfide-bridged peptide libraries to (i) efficiently generate large mass spectral reference data for linked peptides at a low cost and (ii) automatically train an algorithm that can efficiently and accurately identify linked peptides from MS/MS spectra. We show that using this approach we were able to identify thousands of MS/MS spectra from disulfide-bridged peptides through comparison with proteome-scale sequence databases and significantly improve the sensitivity of cross-linked peptide identification. This allowed us to identify 60% more direct pairwise interactions between the protein subunits in the 20S proteasome complex than existing tools on cross-linking studies of the proteasome complexes. The basic framework of this approach and the MS/MS reference dataset generated should be valuable resources for the future development of new tools for the identification of linked peptides. PMID:24493012

  20. Combined Statistical Analyses of Peptide Intensities and Peptide Occurrences Improves Identification of Significant Peptides from MS-based Proteomics Data

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.; McCue, Lee Ann; Waters, Katrina M.; Matzke, Melissa M.; Jacobs, Jon M.; Metz, Thomas O.; Varnum, Susan M.; Pounds, Joel G.

    2010-11-01

    Liquid chromatography-mass spectrometry-based (LC-MS) proteomics uses peak intensities of proteolytic peptides to infer the differential abundance of peptides/proteins. However, substantial run-to-run variability in peptide intensities and observations (presence/absence) of peptides makes data analysis quite challenging. The missing abundance values in LC-MS proteomics data are difficult to address with traditional imputation-based approaches because the mechanisms by which data are missing are unknown a priori. Data can be missing due to random mechanisms such as experimental error, or non-random mechanisms such as a true biological effect. We present a statistical approach that uses a test of independence known as a G-test to test the null hypothesis of independence between the number of missing values and the experimental groups. We pair the G-test results evaluating independence of missing data (IMD) with a standard analysis of variance (ANOVA) that uses only means and variances computed from the observed data. Each peptide is therefore represented by two statistical confidence metrics, one for qualitative differential observation and one for quantitative differential intensity. We use two simulated and two real LC-MS datasets to demonstrate the robustness and sensitivity of the ANOVA-IMD approach for assigning confidence to peptides with significant differential abundance among experimental groups.

  1. On comparison of SimTandem with state-of-the-art peptide identification tools, efficiency of precursor mass filter and dealing with variable modifications.

    PubMed

    Novák, Jiří; Sachsenberg, Timo; Hoksza, David; Skopal, Tomáš; Kohlbacher, Oliver

    2013-11-14

    The similarity search in theoretical mass spectra generated from protein sequence databases is a widely accepted approach for identification of peptides from query mass spectra produced by shotgun proteomics. Growing protein sequence databases and noisy query spectra demand database indexing techniques and better similarity measures for the comparison of theoretical spectra against query spectra. We employ a modification of previously proposed parameterized Hausdorff distance for comparisons of mass spectra. The new distance outperforms the original distance, the angle distance and state-of-the-art peptide identification tools OMSSA and X!Tandem in the number of identified peptides even though the q-value is only 0.001. When a precursor mass filter is used as a database indexing technique, our method outperforms OMSSA in the speed of search. When variable modifications are not searched, the search time is similar to X!Tandem. We show that the precursor mass filter is an efficient database indexing technique for high-accuracy data even though many variable modifications are being searched. We demonstrate that the number of identified peptides is bigger when variable modifications are searched separately by more search runs of a peptide identification engine. Otherwise, the false discovery rates are affected by mixing unmodified and modified spectra together resulting in a lower number of identified peptides. Our method is implemented in the freely available application SimTandem which can be used in the framework TOPP based on OpenMS.

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

    PubMed

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

    2011-08-01

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

  3. MSblender: A probabilistic approach for integrating peptide identifications from multiple database search engines.

    PubMed

    Kwon, Taejoon; Choi, Hyungwon; Vogel, Christine; Nesvizhskii, Alexey I; Marcotte, Edward M

    2011-07-01

    Shotgun proteomics using mass spectrometry is a powerful method for protein identification but suffers limited sensitivity in complex samples. Integrating peptide identifications from multiple database search engines is a promising strategy to increase the number of peptide identifications and reduce the volume of unassigned tandem mass spectra. Existing methods pool statistical significance scores such as p-values or posterior probabilities of peptide-spectrum matches (PSMs) from multiple search engines after high scoring peptides have been assigned to spectra, but these methods lack reliable control of identification error rates as data are integrated from different search engines. We developed a statistically coherent method for integrative analysis, termed MSblender. MSblender converts raw search scores from search engines into a probability score for every possible PSM and properly accounts for the correlation between search scores. The method reliably estimates false discovery rates and identifies more PSMs than any single search engine at the same false discovery rate. Increased identifications increment spectral counts for most proteins and allow quantification of proteins that would not have been quantified by individual search engines. We also demonstrate that enhanced quantification contributes to improve sensitivity in differential expression analyses.

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

    PubMed

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

    2011-08-15

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

  5. DHPC strongly affects the structure and oligomerization propensity of Alzheimer's Aβ(1-40) peptide.

    PubMed

    Dahse, Kirsten; Garvey, Megan; Kovermann, Michael; Vogel, Alexander; Balbach, Jochen; Fändrich, Marcus; Fahr, Alfred

    2010-11-01

    Alzheimer's disease (AD) is thought to depend on the deleterious action of amyloid fibrils or oligomers derived from β-amyloid (Aβ) peptide. Out of various known Aβ alloforms, the 40-residue peptide Aβ(1-40) occurs at highest concentrations inside the brains of AD patients. Its aggregation properties critically depend on lipids, and it was thus proposed that lipids could play a major role in AD. To better understand their possible effects on the structure of Aβ and on the ability of this peptide to form potentially detrimental amyloid structures, we here analyze the interactions between Aβ(1-40) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). DHPC has served, due to its controlled properties, as a major model system for studying general lipid properties. Here, we show that DHPC concentrations of 8 mM or higher exert dramatic effects on the conformation of soluble Aβ(1-40) peptide and induce the formation of β-sheet structure at high levels. By contrast, we find that DHPC concentrations well below the critical micelle concentration present no discernible effect on the conformation of soluble Aβ, although they substantially affect the peptide's oligomerization and fibrillation kinetics. These data imply that subtle lipid-peptide interactions suffice in controlling the overall aggregation properties and drastically accelerate, or delay, the fibrillation kinetics of Aβ peptide in near-physiological buffer solutions.

  6. Matching cross-linked peptide spectra: only as good as the worse identification.

    PubMed

    Trnka, Michael J; Baker, Peter R; Robinson, Philip J J; Burlingame, A L; Chalkley, Robert J

    2014-02-01

    Chemical cross-linking mass spectrometry identifies interacting surfaces within a protein assembly through labeling with bifunctional reagents and identifying the covalently modified peptides. These yield distance constraints that provide a powerful means to model the three-dimensional structure of the assembly. Bioinformatic analysis of cross-linked data resulting from large protein assemblies is challenging because each cross-linked product contains two covalently linked peptides, each of which must be correctly identified from a complex matrix of potential confounders. Protein Prospector addresses these issues through a complementary mass modification strategy in which each peptide is searched and identified separately. We demonstrate this strategy with an analysis of RNA polymerase II. False discovery rates (FDRs) are assessed via comparison of cross-linking data to crystal structure, as well as by using a decoy database strategy. Parameters that are most useful for positive identification of cross-linked spectra are explored. We find that fragmentation spectra generally contain more product ions from one of the two peptides constituting the cross-link. Hence, metrics reflecting the quality of the spectral match to the less confident peptide provide the most discriminatory power between correct and incorrect matches. A support vector machine model was built to further improve classification of cross-linked peptide hits. Furthermore, the frequency with which peptides cross-linked via common acylating reagents fragment to produce diagnostic, cross-linker-specific ions is assessed. The threshold for successful identification of the cross-linked peptide product depends upon the complexity of the sample under investigation. Protein Prospector, by focusing the reliability assessment on the least confident peptide, is better able to control the FDR for results as larger complexes and databases are analyzed. In addition, when FDR thresholds are calculated separately

  7. Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification.

    PubMed

    Guixer, B; Arroyo, X; Belda, I; Sabidó, E; Teixidó, M; Giralt, E

    2016-09-01

    The blood-brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma-targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high-throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d-amino acids, this approach screens only protease-resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state-of-the-art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix-and-split technique to generate a library based on the following: Ac-d-Arg-XXXXX-NH2 , where X were: d-Ala (a), d-Arg (r), d-Ile (i), d-Glu (e), d-Ser (s), d-Trp (w) or d-Pro (p). The assays used comprised the in vitro cell-based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two-step mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease-resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID

  8. How Prior Knowledge Affects Word Identification and Comprehension

    ERIC Educational Resources Information Center

    Priebe, Sarah J.; Keenan, Janice M.; Miller, Amanda C.

    2012-01-01

    While prior knowledge of a passage topic is known to facilitate comprehension, little is known about how it affects word identification. We examined oral reading errors in good and poor readers when reading a passage where they either had prior knowledge of the passage topic or did not. Children who had prior knowledge of the topic were matched on…

  9. Statistical model for large-scale peptide identification in databases from tandem mass spectra using SEQUEST.

    PubMed

    López-Ferrer, Daniel; Martínez-Bartolomé, Salvador; Villar, Margarita; Campillos, Mónica; Martín-Maroto, Fernando; Vázquez, Jesús

    2004-12-01

    Recent technological advances have made multidimensional peptide separation techniques coupled with tandem mass spectrometry the method of choice for high-throughput identification of proteins. Due to these advances, the development of software tools for large-scale, fully automated, unambiguous peptide identification is highly necessary. In this work, we have used as a model the nuclear proteome from Jurkat cells and present a processing algorithm that allows accurate predictions of random matching distributions, based on the two SEQUEST scores Xcorr and DeltaCn. Our method permits a very simple and precise calculation of the probabilities associated with individual peptide assignments, as well as of the false discovery rate among the peptides identified in any experiment. A further mathematical analysis demonstrates that the score distributions are highly dependent on database size and precursor mass window and suggests that the probability associated with SEQUEST scores depends on the number of candidate peptide sequences available for the search. Our results highlight the importance of adjusting the filtering criteria to discriminate between correct and incorrect peptide sequences according to the circumstances of each particular experiment.

  10. Peptide identification via constrained multi-objective optimization: Pareto-based genetic algorithms

    SciTech Connect

    Malard, Joel M.; Heredia-Langner, Alejandro; Cannon, William R.; Mooney, Ryan W.; Baxter, Douglas J.

    2005-12-10

    Automatic data-base independent peptide identification from collision-induced dissociation tandem mass spectrometry data is made difficult by large plateaus in the fitness landscapes of scoring functions and the fuzzy nature of the constraints that is due to noise in the data. Two different scoring functions are combined into a parallel multi-objective optimization framework.

  11. High-throughput identification of putative receptors for cancer-binding peptides using biopanning and microarray analysis

    PubMed Central

    Ferraro, Daniel J; Bhave, Sandeep R; Kotipatruni, Rama P; Hunn, Jeremy C; Wildman, Scott A; Hong, Charles; Dadey, David Y. A.; Muhoro, Lincoln K.; Jaboin, Jerry J; Thotala, Dinesh; Hallahan, Dennis E

    2013-01-01

    Phage-display peptide biopanning has been successfully used to identify cancer-targeting peptides in multiple models. For cancer-binding peptides, identification of the peptide receptor is necessary to demonstrate mechanism of action and to further optimize specificity and target binding. The process of receptor identification can be slow and some peptides may turn out to bind ubiquitous proteins not suitable for further drug development. In this report, we describe a high-throughput method for screening a large number of peptides in parallel to identify peptide receptors, which we have termed “reverse biopanning,” which can then be selected for further development based on their peptide receptor. To demonstrate this method, we screened a library of 39 peptides previously identified in our laboratory to bind specifically cancers after irradiation. The reverse biopanning process identified 2 peptides, RKFLMTTRYSRV and KTAKKNVFFCSV, as candidate ligands for the protein tax interacting protein 1 (TIP-1), a protein previously identified in our laboratory to be expressed in the cell surface in tumors and upregulated after exposure to ionizing radiation. We used computational modeling as the initial method for rapid validation of peptide-TIP-1 binding. Pseudo-binding energies were calculated to be −360.645 kcal/mol, −487.239 kcal/mol, and −595.328 kcal/mol for HVGGSSV, TTRYSRV, and NVFFCSV respectively, suggesting that the peptides would have at least similar, if not stronger, binding to TIP-1 compared to the known TIP-1 binding peptide HVGGSSV. We validated peptide in vitro via electrophoretic mobility shift assay, which showed strong binding of RKFLMTTRYSRV and the truncated form TTRYSRV. This method allows for the identification of many peptide receptors and subsequent selection of peptides for further drug development based on the peptide receptor. PMID:23147990

  12. Identification of bitter peptides in aged cheddar cheese.

    PubMed

    Karametsi, Konstantinia; Kokkinidou, Smaro; Ronningen, Ian; Peterson, Devin G

    2014-08-13

    The compounds responsible for the bitter taste of aged "sharp" Cheddar cheese were characterized. Sensory-guided fractionation techniques using gel permeation chromatography and multi-dimension semi-preparative reversed-phase high-performance liquid chromatography revealed the presence of multiple bitter compounds. The compounds with the highest perceived bitterness intensity were identified by tandem mass spectrometry de novo peptide sequencing as GPVRGPFPIIV, YQEPVLGPVRGPFPI, MPFPKYPVEP, MAPKHKEMPFPKYPVEPF, and APHGKEMPFPKYPVEPF; all originated from β-casein. Subsequent quantitative liquid chromatography-tandem mass spectrometry analysis reported that the concentrations of GPVRGPFPIIV, YQEPVLGPVRGPFPI, and MPFPKYPVEP increased during maturation by 28.7-, 3.1-, and 1.8-fold, respectively. When directly compared to young "mild" Cheddar, APHGKEMPFPKYPVEPF was reported only in the sharp Cheddar cheese, whereas the concentration of MAPKHKEMPFPKYPVEPF did not change. Further taste re-engineering sensory experiments confirmed the importance of the identified peptides to the bitterness of sharp Cheddar. The bitter intensity of the aged "sharp" Cheddar model (mild Cheddar with equivalent concentrations of the five bitter peptides in the sharp sample) was rated as not significantly different from the authentic sharp Cheddar cheese. Among the five peptides, GPVRGPFPIIV was reported to be the main contributor to the bitterness intensity of sharp Cheddar. Furthermore, a difference from control sensory test also confirmed the significance of the bitter taste to the overall perception of aged Cheddar flavor. The sharp Cheddar model was reported to be significantly more similar to aged "sharp" Cheddar in comparison to the young "mild" Cheddar cheese sample. PMID:25075877

  13. Identification of Microcystis aeruginosa Peptides Responsible for Allergic Sensitization and Characterization of Functional Interactions between Cyanobacterial Toxins and Immunogenic Peptides

    PubMed Central

    Geh, Esmond N.; Ghosh, Debajyoti; McKell, Melanie; de la Cruz, Armah A.; Stelma, Gerard

    2015-01-01

    . Identification of Microcystis aeruginosa peptides responsible for allergic sensitization and characterization of functional interactions between cyanobacterial toxins and immunogenic peptides. Environ Health Perspect 123:1159–1166; http://dx.doi.org/10.1289/ehp.1409065 PMID:25902363

  14. Overcoming species boundaries in peptide identification with Bayesian information criterion-driven error-tolerant peptide search (BICEPS).

    PubMed

    Renard, Bernhard Y; Xu, Buote; Kirchner, Marc; Zickmann, Franziska; Winter, Dominic; Korten, Simone; Brattig, Norbert W; Tzur, Amit; Hamprecht, Fred A; Steen, Hanno

    2012-07-01

    Currently, the reliable identification of peptides and proteins is only feasible when thoroughly annotated sequence databases are available. Although sequencing capacities continue to grow, many organisms remain without reliable, fully annotated reference genomes required for proteomic analyses. Standard database search algorithms fail to identify peptides that are not exactly contained in a protein database. De novo searches are generally hindered by their restricted reliability, and current error-tolerant search strategies are limited by global, heuristic tradeoffs between database and spectral information. We propose a Bayesian information criterion-driven error-tolerant peptide search (BICEPS) and offer an open source implementation based on this statistical criterion to automatically balance the information of each single spectrum and the database, while limiting the run time. We show that BICEPS performs as well as current database search algorithms when such algorithms are applied to sequenced organisms, whereas BICEPS only uses a remotely related organism database. For instance, we use a chicken instead of a human database corresponding to an evolutionary distance of more than 300 million years (International Chicken Genome Sequencing Consortium (2004) Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695-716). We demonstrate the successful application to cross-species proteomics with a 33% increase in the number of identified proteins for a filarial nematode sample of Litomosoides sigmodontis.

  15. Overcoming Species Boundaries in Peptide Identification with Bayesian Information Criterion-driven Error-tolerant Peptide Search (BICEPS)*

    PubMed Central

    Renard, Bernhard Y.; Xu, Buote; Kirchner, Marc; Zickmann, Franziska; Winter, Dominic; Korten, Simone; Brattig, Norbert W.; Tzur, Amit; Hamprecht, Fred A.; Steen, Hanno

    2012-01-01

    Currently, the reliable identification of peptides and proteins is only feasible when thoroughly annotated sequence databases are available. Although sequencing capacities continue to grow, many organisms remain without reliable, fully annotated reference genomes required for proteomic analyses. Standard database search algorithms fail to identify peptides that are not exactly contained in a protein database. De novo searches are generally hindered by their restricted reliability, and current error-tolerant search strategies are limited by global, heuristic tradeoffs between database and spectral information. We propose a Bayesian information criterion-driven error-tolerant peptide search (BICEPS) and offer an open source implementation based on this statistical criterion to automatically balance the information of each single spectrum and the database, while limiting the run time. We show that BICEPS performs as well as current database search algorithms when such algorithms are applied to sequenced organisms, whereas BICEPS only uses a remotely related organism database. For instance, we use a chicken instead of a human database corresponding to an evolutionary distance of more than 300 million years (International Chicken Genome Sequencing Consortium (2004) Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695–716). We demonstrate the successful application to cross-species proteomics with a 33% increase in the number of identified proteins for a filarial nematode sample of Litomosoides sigmodontis. PMID:22493179

  16. Molecular identification of Malassezia species isolated from dermatitis affections.

    PubMed

    Affes, M; Ben Salah, S; Makni, F; Sellami, H; Ayadi, A

    2009-05-01

    The lipophilic yeast of the genus Malassezia are opportunistic microorganisms of the skin microflora but they can be agents of various dermatomycoses. The aim of this study was to perform molecular identification of the commonly isolated Malassezia species from various dermatomycoses in our region. Thirty strains of Malassezia were isolated from different dermatologic affections: pityriasis versicolor (17), dandruff (5), seborrheic dermatitis (4), onyxis (2), folliculitis (1) and blepharitis (1). These species were identified by their morphological features and biochemical characterisation. The molecular identification was achieved by amplification of the internal transcribed spacer region by simple PCR. PCR technique was used for molecular characterisation of four Malassezia species: Malassezia globosa (270 bp), Malassezia furfur (230 bp), Malassezia sympodialis (190 bp) and Malassezia restricta (320 bp). We have detected the association between M. furfur and M. sympodialis in 16% and confirmed presumptive identification in 70% of the cases. The phenotypic identification based on microscopic and physiological method is difficult and time consuming. The application of a simple PCR method provides a sensitive and rapid identification system for Malassezia species, which may be applied in epidemiological surveys and routine practice. PMID:18643889

  17. A peptide nanofibrous indicator for eye-detectable cancer cell identification.

    PubMed

    Chen, Chang-Sheng; Xu, Xiao-Ding; Wang, Ya; Yang, Juan; Jia, Hui-Zhen; Cheng, Han; Chu, Chih-Chang; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-03-25

    A unique peptide nanofibrous indicator (NFI) is fabricated by mixing a borono-peptide with alizarin red S, followed by subsequent binding and self-assembly. The NFI thus obtained exhibits an intense response to sialyl Lewis X tetrasaccharide, which is overexpressed in human hepatocellular carcinoma cell lines. Importantly, this NFI has the capability of specifically recognizing human hepatocellular liver carcinoma (HepG2) cells through the eye-detectable color change resulting from strong binding-induced displacement. This novel technique for cancer cell identification through direct unaided eye judgment will open up an innovative platform for cancer cell detection.

  18. A peptide nanofibrous indicator for eye-detectable cancer cell identification.

    PubMed

    Chen, Chang-Sheng; Xu, Xiao-Ding; Wang, Ya; Yang, Juan; Jia, Hui-Zhen; Cheng, Han; Chu, Chih-Chang; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-03-25

    A unique peptide nanofibrous indicator (NFI) is fabricated by mixing a borono-peptide with alizarin red S, followed by subsequent binding and self-assembly. The NFI thus obtained exhibits an intense response to sialyl Lewis X tetrasaccharide, which is overexpressed in human hepatocellular carcinoma cell lines. Importantly, this NFI has the capability of specifically recognizing human hepatocellular liver carcinoma (HepG2) cells through the eye-detectable color change resulting from strong binding-induced displacement. This novel technique for cancer cell identification through direct unaided eye judgment will open up an innovative platform for cancer cell detection. PMID:23225693

  19. Immunocytochemical and molecular data guide peptide identification by mass spectrometry: orcokinin and orcomyotropin-related peptides in the stomatogastric nervous system of several crustacean species.

    PubMed

    Skiebe, P; Dreger, M; Börner, J; Meseke, M; Weckwerth, W

    2003-07-01

    In order to identify new orcokinin and orcomyotropin-related peptides in crustaceans, molecular and immunocytochemical data were combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the crayfish Procambarus clarkii, four orcokinins and an orcomyotropin-related peptide are present on the precursor. Because these peptides are highly conserved, we assumed that other species have an identical number of peptides. To identify the peptides, immunocytochemistry was used to localize the regions of the stomatogastric nervous system in which orcokinins are predominantly present. One of the regions predominantly containing orcokinins was a previously undescribed olive-shaped neuropil region within the commissural ganglia of the lobsters Homarus americanus and Homarus gammarus. MALDI-TOF MS on these regions identified peptide masses that always occur together with the known orcokinins. Seven peptide ions occurred together in the peptide massspectra of the lobsters. Mass spectrometric fragmentation by MALDI-MS post-source decay (PSD) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI Q-TOF MS) collision-induced dissociation (CID) were used in the identification of six of these masses, either as orcokinins or as orcomyotropin-related peptides and revealed three hitherto unknown peptide variants, two of which are [His13]-orcokinin ([M+H]+ = 1540.8 Da) and an orcomyotropin-related peptide FDAFTTGFGHN ([M+H]+ = 1213.5 Da). The mass of the third previously unknown orcokinin variant corresponded to that of an identified orcokinin, but PSD fragmentation did not support the suggested amino acid sequence. CID analysis allowed partial de novo sequencing of this peptide. In the crab Cancer pagurus, five orcokinins and an orcomyotropin-related peptide were unambigously identified, including the previously unknown peptide variant [Ser9-Val13]-orcokinin ([M+H]+ = 1532.8 Da). PMID:14528921

  20. Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea.

    PubMed

    Ong, Ta-Hsuan; Romanova, Elena V; Roberts-Galbraith, Rachel H; Yang, Ning; Zimmerman, Tyler A; Collins, James J; Lee, Ji Eun; Kelleher, Neil L; Newmark, Phillip A; Sweedler, Jonathan V

    2016-04-01

    Tissue regeneration is a complex process that involves a mosaic of molecules that vary spatially and temporally. Insights into the chemical signaling underlying this process can be achieved with a multiplex and untargeted chemical imaging method such as mass spectrometry imaging (MSI), which can enablede novostudies of nervous system regeneration. A combination of MSI and multivariate statistics was used to differentiate peptide dynamics in the freshwater planarian flatwormSchmidtea mediterraneaat different time points during cephalic ganglia regeneration. A protocol was developed to makeS. mediterraneatissues amenable for MSI. MS ion images of planarian tissue sections allow changes in peptides and unknown compounds to be followed as a function of cephalic ganglia regeneration. In conjunction with fluorescence imaging, our results suggest that even though the cephalic ganglia structure is visible after 6 days of regeneration, the original chemical composition of these regenerated structures is regained only after 12 days. Differences were observed in many peptides, such as those derived from secreted peptide 4 and EYE53-1. Peptidomic analysis further identified multiple peptides from various known prohormones, histone proteins, and DNA- and RNA-binding proteins as being associated with the regeneration process. Mass spectrometry data also facilitated the identification of a new prohormone, which we have named secreted peptide prohormone 20 (SPP-20), and is up-regulated during regeneration in planarians.

  1. Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea*

    PubMed Central

    Ong, Ta-Hsuan; Romanova, Elena V.; Roberts-Galbraith, Rachel H.; Yang, Ning; Zimmerman, Tyler A.; Collins, James J.; Lee, Ji Eun; Kelleher, Neil L.; Newmark, Phillip A.; Sweedler, Jonathan V.

    2016-01-01

    Tissue regeneration is a complex process that involves a mosaic of molecules that vary spatially and temporally. Insights into the chemical signaling underlying this process can be achieved with a multiplex and untargeted chemical imaging method such as mass spectrometry imaging (MSI), which can enable de novo studies of nervous system regeneration. A combination of MSI and multivariate statistics was used to differentiate peptide dynamics in the freshwater planarian flatworm Schmidtea mediterranea at different time points during cephalic ganglia regeneration. A protocol was developed to make S. mediterranea tissues amenable for MSI. MS ion images of planarian tissue sections allow changes in peptides and unknown compounds to be followed as a function of cephalic ganglia regeneration. In conjunction with fluorescence imaging, our results suggest that even though the cephalic ganglia structure is visible after 6 days of regeneration, the original chemical composition of these regenerated structures is regained only after 12 days. Differences were observed in many peptides, such as those derived from secreted peptide 4 and EYE53-1. Peptidomic analysis further identified multiple peptides from various known prohormones, histone proteins, and DNA- and RNA-binding proteins as being associated with the regeneration process. Mass spectrometry data also facilitated the identification of a new prohormone, which we have named secreted peptide prohormone 20 (SPP-20), and is up-regulated during regeneration in planarians. PMID:26884331

  2. Epitope Identification from Fixed-complexity Random-sequence Peptide Microarrays

    PubMed Central

    Richer, Josh; Johnston, Stephen Albert; Stafford, Phillip

    2015-01-01

    Antibodies play an important role in modern science and medicine. They are essential in many biological assays and have emerged as an important class of therapeutics. Unfortunately, current methods for mapping antibody epitopes require costly synthesis or enrichment steps, and no low-cost universal platform exists. In order to address this, we tested a random-sequence peptide microarray consisting of over 330,000 unique peptide sequences sampling 83% of all possible tetramers and 27% of pentamers. It is a single, unbiased platform that can be used in many different types of tests, it does not rely on informatic selection of peptides for a particular proteome, and it does not require iterative rounds of selection. In order to optimize the platform, we developed an algorithm that considers the significance of k-length peptide subsequences (k-mers) within selected peptides that come from the microarray. We tested eight monoclonal antibodies and seven infectious disease cohorts. The method correctly identified five of the eight monoclonal epitopes and identified both reported and unreported epitope candidates in the infectious disease cohorts. This algorithm could greatly enhance the utility of random-sequence peptide microarrays by enabling rapid epitope mapping and antigen identification. PMID:25368412

  3. Target Promiscuity and Heterogeneous Effects of Tarantula Venom Peptides Affecting Na+ and K+ Ion Channels*

    PubMed Central

    Redaelli, Elisa; Cassulini, Rita Restano; Silva, Deyanira Fuentes; Clement, Herlinda; Schiavon, Emanuele; Zamudio, Fernando Z.; Odell, George; Arcangeli, Annarosa; Clare, Jeffrey J.; Alagón, Alejandro; de la Vega, Ricardo C. Rodríguez; Possani, Lourival D.; Wanke, Enzo

    2010-01-01

    Venom-derived peptide modulators of ion channel gating are regarded as essential tools for understanding the molecular motions that occur during the opening and closing of ion channels. In this study, we present the characterization of five spider toxins on 12 human voltage-gated ion channels, following observations about the target promiscuity of some spider toxins and the ongoing revision of their “canonical” gating-modifying mode of action. The peptides were purified de novo from the venom of Grammostola rosea tarantulas, and their sequences were confirmed by Edman degradation and mass spectrometry analysis. Their effects on seven tetrodotoxin-sensitive Na+ channels, the three human ether-à-go-go (hERG)-related K+ channels, and two human Shaker-related K+ channels were extensively characterized by electrophysiological techniques. All the peptides inhibited ion conduction through all the Na+ channels tested, although with distinctive patterns. The peptides also affected the three pharmaceutically relevant hERG isoforms differently. At higher concentrations, all peptides also modified the gating of the Na+ channels by shifting the activation to more positive potentials, whereas more complex effects were recorded on hERG channels. No effects were evident on the two Shaker-related K+ channels at concentrations well above the IC50 value for the affected channels. Given the sequence diversity of the tested peptides, we propose that tarantula toxins should be considered both as multimode and target-promiscuous ion channel modulators; both features should not be ignored when extracting mechanistic interpretations about ion channel gating. Our observations could also aid in future structure-function studies and might help the development of novel ion channel-specific drugs. PMID:19955179

  4. Does post-identification feedback affect evaluations of eyewitness testimony and identification procedures?

    PubMed

    Douglass, Amy Bradfield; Neuschatz, Jeffrey S; Imrich, Jennifer; Wilkinson, Miranda

    2010-08-01

    Two experiments were conducted to test whether post-identification feedback affects evaluations of eyewitnesses. In Experiment 1 (N = 156), evaluators viewed eyewitness testimony. They evaluated witnesses who received confirming post-identification feedback as more accurate and more confident, among other judgments, compared with witnesses who received disconfirming post-identification feedback or no feedback. This pattern persisted regardless of whether the witness's confidence statement was included in the testimony. In Experiment 2 (N = 161), witness evaluators viewed the actual identification procedure in which feedback was delivered. Instructions to disregard the feedback were manipulated. Again, witnesses who received confirming feedback were assessed more positively. This pattern occurred even when witness evaluators received instructions to disregard the feedback. These experiments are the first to confirm researchers' assumptions that feedback effects on witnesses translate to changes in judgments of those witnesses. PMID:19585229

  5. How Prior Knowledge Affects Word Identification and Comprehension

    PubMed Central

    Priebe, Sarah J.; Keenan, Janice M.; Miller, Amanda C.

    2011-01-01

    While prior knowledge of a passage topic is known to facilitate comprehension, little is known about how it affects word identification. We examined oral reading errors in good and poor readers when reading a passage where they either had prior knowledge of the passage topic or did not. Children who had prior knowledge of the topic were matched on decoding skill to children who did not know the topic so that the groups differed only on knowledge of the passage topic. Prior knowledge of the passage topic was found to significantly increase fluency and reduce reading errors, especially errors based on graphic information, in poor readers. Two possible mechanisms of how prior knowledge might operate to facilitate word identification were evaluated using the pattern of error types, as was the relationship of errors to comprehension. Implications of knowledge effects for assessment and educational policy are discussed. PMID:21799586

  6. A chirality change in XPC- and Sfi1-derived peptides affects their affinity for centrin.

    PubMed

    Grecu, Dora; Irudayaraj, Victor Paul Raj; Martinez-Sanz, Juan; Mallet, Jean-Maurice; Assairi, Liliane

    2016-04-01

    The Ca(2+)-binding protein centrin binds to a hydrophobic motif (W(1)xxL(4)xxxL(8)) included in the sequence of several cellular targets: XPC (xeroderma pigmentosum group C protein), Sfi1 (suppressor of fermentation-induced loss of stress resistance protein1), and Sac3 [the central component of the transcription and mRNA export (TREX-2) complex]. However, centrin binding occurs in a reversed orientation (L(8)xxxL(4)xxW(1)) for Sfi1 and Sac3 compared with XPC. Because D-peptides have been investigated for future therapeutic use, we analyzed their centrin-binding properties. Their affinity for centrin was measured using isothermal titration calorimetry. The chirality change in the target-derived peptides affected their ability to bind centrin in a specific manner depending on the sequence orientation of the centrin-binding motif. In contrast to L-XPC-P10, D-XPC-P10 bound C-HsCen1 in a Ca(2+)-dependent manner and to a lesser extent. D-XPC-P10 exhibited a reduced affinity for C-HsCen1 (Ka=0.064 × 10(6) M(-1)) by a factor of 2000 compared with L-XPC-P10 (Ka=132 × 10(6) M(-1)). D-peptides have a lower affinity than L-peptides for centrin, and the strength of this affinity depends on the sequence orientation of the target-derived peptides. The residual affinity observed for D-XPC suggests that the use of d-peptides represents a promising strategy for inhibiting centrin binding to its targets.

  7. Identification of potent 11mer glucagon-like peptide-1 receptor agonist peptides with novel C-terminal amino acids: Homohomophenylalanine analogs.

    PubMed

    Haque, Tasir S; Lee, Ving G; Riexinger, Douglas; Lei, Ming; Malmstrom, Sarah; Xin, Li; Han, Songping; Mapelli, Claudio; Cooper, Christopher B; Zhang, Ge; Ewing, William R; Krupinski, John

    2010-05-01

    We report the identification of potent agonists of the Glucagon-Like Peptide-1 Receptor (GLP-1R). These compounds are short, 11 amino acid peptides containing several unnatural amino acids, including (in particular) analogs of homohomophenylalanine (hhPhe) at the C-terminal position. Typically the functional activity of the more potent peptides in this class is in the low picomolar range in an in vitro cAMP assay, with one example demonstrating excellent in vivo activity in an ob/ob mouse model of diabetes.

  8. Identification of a NFκB inhibitory peptide from tryptic β-casein hydrolysate.

    PubMed

    Malinowski, J; Klempt, M; Clawin-Rädecker, I; Lorenzen, P Chr; Meisel, H

    2014-12-15

    Several bioactive peptides are encrypted within the sequence of major milk proteins, requiring enzymatic proteolysis for release and activation. The present study aimed at the identification of potential anti-inflammatory activities in tryptic hydrolysates of bovine β-casein. Inflammatory processes involve in most cases an activation of Nuclear factor Kappa-light-chain enhancer of activated B cells (NFκB), which is a pro-inflammatory transcription factor of several genes. Hence, a NFκB reporter cell line was established, and TNF-α mediated activation of NFκB was used as a measurement. Bovine β-casein (β-CN) was hydrolysed by trypsin and fractionated by ultrafiltration. Total proteolysate as well as the fraction containing peptides between 1 and 5 kDa showed an inhibitory effect in the cell-based assay, while the fraction containing molecules smaller than 1 kDa did not. This anti-inflammatory effect was ascribed to a group of large, hydrophobic peptides, which were identified using LC-MS. The main peptide was synthesised and showed a significant anti-inflammatory effect in HEK(nfkb-RE)-cells. Thus, for the first time, a casein-derived peptide having an anti-inflammatory effect in vitro has been identified.

  9. Data-independent-acquisition mass spectrometry for identification of targeted-peptide site-specific modifications.

    PubMed

    Porter, Caleb J; Bereman, Michael S

    2015-09-01

    We present a novel strategy based on data-independent acquisition coupled to targeted data extraction for the detection and identification of site-specific modifications of targeted peptides in a completely unbiased manner. This method requires prior knowledge of the site of the modification along the peptide backbone from the protein of interest, but not the mass of the modification. The procedure, named multiplex adduct peptide profiling (MAPP), consists of three steps: 1) A fragment-ion tag is extracted from the data, consisting of the b-type and y-type ion series from the N and C-terminus, respectively, up to the amino-acid position that is believed to be modified; 2) MS1 features are matched to the fragment-ion tag in retention-time space, using the isolation window as a pre-filter to enable calculation of the mass of the modification; and 3) modified fragment ions are overlaid with the unmodified fragment ions to verify the mass calculated in step 2. We discuss the development, applications, and limitations of this new method for detection of unknown peptide modifications. We present an application of the method in profiling adducted peptides derived from abundant proteins in biological fluids with the ultimate objective of detecting biomarkers of exposure to reactive species.

  10. BAGEL3: automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides

    PubMed Central

    van Heel, Auke J.; de Jong, Anne; Montalbán-López, Manuel; Kok, Jan; Kuipers, Oscar P.

    2013-01-01

    Identifying genes encoding bacteriocins and ribosomally synthesized and posttranslationally modified peptides (RiPPs) can be a challenging task. Especially those peptides that do not have strong homology to previously identified peptides can easily be overlooked. Extensive use of BAGEL2 and user feedback has led us to develop BAGEL3. BAGEL3 features genome mining of prokaryotes, which is largely independent of open reading frame (ORF) predictions and has been extended to cover more (novel) classes of posttranslationally modified peptides. BAGEL3 uses an identification approach that combines direct mining for the gene and indirect mining via context genes. Especially for heavily modified peptides like lanthipeptides, sactipeptides, glycocins and others, this genetic context harbors valuable information that is used for mining purposes. The bacteriocin and context protein databases have been updated and it is now easy for users to submit novel bacteriocins or RiPPs. The output has been simplified to allow user-friendly analysis of the results, in particular for large (meta-genomic) datasets. The genetic context of identified candidate genes is fully annotated. As input, BAGEL3 uses FASTA DNA sequences or folders containing multiple FASTA formatted files. BAGEL3 is freely accessible at http://bagel.molgenrug.nl. PMID:23677608

  11. Identification of small peptides arising from hydrolysis of meat proteins in dry fermented sausages.

    PubMed

    López, Constanza M; Bru, Elena; Vignolo, Graciela M; Fadda, Silvina G

    2015-06-01

    In this study, proteolysis and low molecular weight (LMW) peptides (<3kDa) from commercial Argentinean fermented sausages were characterized by applying a peptidomic approach. Protein profiles and peptides obtained by Tricine-SDS-PAGE and RP-HPLC-MS, respectively, allowed distinguishing two different types of fermented sausages, although no specific biomarkers relating to commercial brands or quality were recognized. From electrophoresis, α-actin, myoglobin, creatine kinase M-type and L-lactate dehydrogenase were degraded at different intensities. In addition, a partial characterization of fermented sausage peptidome through the identification of 36 peptides, in the range of 1000-2100 Da, arising from sarcoplasmic (28) and myofibrillar (8) proteins was achieved. These peptides had been originated from α-actin, myoglobin, and creatine kinase M-type, but also from the hydrolysis of other proteins not previously reported. Although muscle enzymes exerted a major role on peptidogenesis, microbial contribution cannot be excluded as it was postulated herein. This work represents a first peptidomic approach for fermented sausages, thereby providing a baseline to define key peptides acting as potential biomarkers.

  12. Identification of Drosophila Mutants Affecting Defense to an Entomopathogenic Fungus

    PubMed Central

    Lu, Hsiao-Ling; Wang, Jonathan B.; Brown, Markus A.; Euerle, Christopher; St. Leger, Raymond J.

    2015-01-01

    Fungi cause the majority of insect disease. However, to date attempts to model host–fungal interactions with Drosophila have focused on opportunistic human pathogens. Here, we performed a screen of 2,613 mutant Drosophila lines to identify host genes affecting susceptibility to the natural insect pathogen Metarhizium anisopliae (Ma549). Overall, 241 (9.22%) mutant lines had altered resistance to Ma549. Life spans ranged from 3.0 to 6.2 days, with females being more susceptible than males in all lines. Speed of kill correlated with within-host growth and onset of sporulation, but total spore production is decoupled from host genotypes. Results showed that mutations affected the ability of Drosophila to restrain rather than tolerate infections and suggested trade-offs between antifungal and antibacterial genes affecting cuticle and gut structural barriers. Approximately, 13% of mutations where in genes previously associated with host pathogen interactions. These encoded fast-acting immune responses including coagulation, phagocytosis, encapsulation and melanization but not the slow-response induction of anti-fungal peptides. The non-immune genes impact a wide variety of biological functions, including behavioral traits. Many have human orthologs already implicated in human disorders; while others were mutations in protein and non-protein coding genes for which disease resistance was the first biological annotation. PMID:26202798

  13. Expression analysis and identification of antimicrobial peptide transcripts from six North American frog species

    USGS Publications Warehouse

    Robertson, Laura S.; Fellers, Gary M.; Marranca, Jamie Marie; Kleeman, Patrick M.

    2013-01-01

    Frogs secrete antimicrobial peptides onto their skin. We describe an assay to preserve and analyze antimicrobial peptide transcripts from field-collected skin secretions that will complement existing methods for peptide analysis. We collected skin secretions from 4 North American species in the field in California and 2 species in the laboratory. Most frogs appeared healthy after release; however, Rana boylii in the Sierra Nevada foothills, but not the Coast Range, showed signs of morbidity and 2 died after handling. The amount of total RNA extracted from skin secretions was higher in R. boylii and R. sierrae compared to R. draytonii, and much higher compared to Pseudacris regilla. Interspecies variation in amount of RNA extracted was not explained by size, but for P. regilla it depended upon collection site and date. RNA extracted from skin secretions from frogs handled with bare hands had poor quality compared to frogs handled with gloves or plastic bags. Thirty-four putative antimicrobial peptide precursor transcripts were identified. This study demonstrates that RNA extracted from skin secretions collected in the field is of high quality suitable for use in sequencing or quantitative PCR (qPCR). However, some species do not secrete profusely, resulting in very little extracted RNA. The ability to measure transcript abundance of antimicrobial peptides in field-collected skin secretions complements proteomic analyses and may provide insight into transcriptional mechanisms that could affect peptide abundance.

  14. How variations in distance affect eyewitness reports and identification accuracy.

    PubMed

    Lindsay, R C L; Semmler, Carolyn; Weber, Nathan; Brewer, Neil; Lindsay, Marilyn R

    2008-12-01

    Witnesses observe crimes at various distances and the courts have to interpret their testimony given the likely quality of witnesses' views of events. We examined how accurately witnesses judged the distance between themselves and a target person, and how distance affected description accuracy, choosing behavior, and identification test accuracy. Over 1,300 participants were approached during normal daily activities, and asked to observe a target person at one of a number of possible distances. Under a Perception, Immediate Memory, or Delayed Memory condition, witnesses provided a brief description of the target, estimated the distance to the target, and then examined a 6-person target-present or target-absent lineup to see if they could identify the target. Errors in distance judgments were often substantial. Description accuracy was mediocre and did not vary systematically with distance. Identification choosing rates were not affected by distance, but decision accuracy declined with distance. Contrary to previous research, a 15-m viewing distance was not critical for discriminating accurate from inaccurate decisions. PMID:18253819

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

    PubMed Central

    2010-01-01

    Background Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i). Results The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set. All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase. Conclusions Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii. PMID:20459704

  16. Identification of distinct physiochemical properties of toxic prefibrillar species formed by A{beta} peptide variants

    SciTech Connect

    Goeransson, Anna-Lena; Nilsson, K. Peter R.; Kagedal, Katarina; Brorsson, Ann-Christin

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer Identification of toxic prefibrillar A{beta} species. Black-Right-Pointing-Pointer Fluorescence measurements using a combined set of fluorophores. Black-Right-Pointing-Pointer Morphology studies using transmission electron microscopy. -- Abstract: The formation of amyloid-{beta} peptide (A{beta}) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of A{beta}. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of A{beta}-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various A{beta} aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those A{beta} peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the A{beta} peptide to form nontoxic versus toxic species.

  17. Signature motif-guided identification of receptors for peptide hormones essential for root meristem growth.

    PubMed

    Song, Wen; Liu, Li; Wang, Jizong; Wu, Zhen; Zhang, Heqiao; Tang, Jiao; Lin, Guangzhong; Wang, Yichuan; Wen, Xing; Li, Wenyang; Han, Zhifu; Guo, Hongwei; Chai, Jijie

    2016-06-01

    Peptide-mediated cell-to-cell signaling has crucial roles in coordination and definition of cellular functions in plants. Peptide-receptor matching is important for understanding the mechanisms underlying peptide-mediated signaling. Here we report the structure-guided identification of root meristem growth factor (RGF) receptors important for plant development. An assay based on a signature ligand recognition motif (Arg-x-Arg) conserved in a subfamily of leucine-rich repeat receptor kinases (LRR-RKs) identified the functionally uncharacterized LRR-RK At4g26540 as a receptor of RGF1 (RGFR1). We further solved the crystal structure of RGF1 in complex with the LRR domain of RGFR1 at a resolution of 2.6 Å, which reveals that the Arg-x-Gly-Gly (RxGG) motif is responsible for specific recognition of the sulfate group of RGF1 by RGFR1. Based on the RxGG motif, we identified additional four RGFRs. Participation of the five RGFRs in RGF-induced signaling is supported by biochemical and genetic data. We also offer evidence showing that SERKs function as co-receptors for RGFs. Taken together, our study identifies RGF receptors and co-receptors that can link RGF signals with their downstream components and provides a proof of principle for structure-based matching of LRR-RKs with their peptide ligands. PMID:27229311

  18. Identification of a Peptide Produced by Bifidobacterium longum CECT 7210 with Antirotaviral Activity

    PubMed Central

    Chenoll, Empar; Casinos, Beatriz; Bataller, Esther; Buesa, Javier; Ramón, Daniel; Genovés, Salvador; Fábrega, Joan; Rivero Urgell, Montserrat; Moreno Muñoz, José A.

    2016-01-01

    Rotavirus is one of the main causes of acute diarrhea and enteritis in infants. Currently, studies are underway to assess the use of probiotics to improve rotavirus vaccine protection. A previous work demonstrated that the probiotic strain Bifidobacterium longum subsp. infantis CECT 7210 is able to hinder rotavirus replication both in vitro and in vivo. The present study takes a systematic approach in order to identify the molecule directly involved in rotavirus inhibition. Supernatant protease digestions revealed both the proteinaceous nature of the active substance and the fact that the molecule responsible for inhibiting rotavirus replication is released to the supernatant. Following purification by cationic exchange chromatography, active fractions were obtained and the functional compound was identified as an 11-amino acid peptide (MHQPHQPLPPT, named 11-mer peptide) with a molecular mass of 1.282 KDa. The functionality of 11-mer was verified using the synthesized peptide in Wa, Ito, and VA70 rotavirus infections of both HT-29 and MA-104 cell lines. Finally, protease activity was detected in B. longum subsp. infantis CECT 7210 supernatant, which releases 11-mer peptide. A preliminary identification of the protease is also included in the study. PMID:27199974

  19. Biochemical Identification of a Linear Cholesterol-Binding Domain within Alzheimer’s β Amyloid Peptide

    PubMed Central

    2012-01-01

    Alzheimer’s β-amyloid (Aβ) peptides can self-organize into amyloid pores that may induce acute neurotoxic effects in brain cells. Membrane cholesterol, which regulates Aβ production and oligomerization, plays a key role in this process. Although several data suggested that cholesterol could bind to Aβ peptides, the molecular mechanisms underlying cholesterol/Aβ interactions are mostly unknown. On the basis of docking studies, we identified the linear fragment 22–35 of Aβ as a potential cholesterol-binding domain. This domain consists of an atypical concatenation of polar/apolar amino acid residues that was not previously found in cholesterol-binding motifs. Using the Langmuir film balance technique, we showed that synthetic peptides Aβ17–40 and Aβ22–35, but not Aβ1–16, could efficiently penetrate into cholesterol monolayers. The interaction between Aβ22–35 and cholesterol was fully saturable and lipid-specific. Single-point mutations of Val-24 and Lys-28 in Aβ22–35 prevented cholesterol binding, whereas mutations at residues 29, 33, and 34 had little to no effect. These data were consistent with the in silico identification of Val-24 and Lys-28 as critical residues for cholesterol binding. We conclude that the linear fragment 22–35 of Aβ is a functional cholesterol-binding domain that could promote the insertion of β-amyloid peptides or amyloid pore formation in cholesterol-rich membrane domains. PMID:23509984

  20. Identification of sites in adenovirus hexon for foreign peptide incorporation.

    PubMed

    Wu, Hongju; Han, Tie; Belousova, Natalya; Krasnykh, Victor; Kashentseva, Elena; Dmitriev, Igor; Kataram, Manjula; Mahasreshti, Parameshwar J; Curiel, David T

    2005-03-01

    Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His6) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His6 epitopes did not appear to mediate His6-dependent viral infection, as assessed in two His6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification. PMID:15731232

  1. Data for identification of GPI-anchored peptides and ω-sites in cancer cell lines

    PubMed Central

    Masuishi, Yusuke; Kimura, Yayoi; Arakawa, Noriaki; Hirano, Hisashi

    2016-01-01

    We present data obtained using a focused proteomics approach to identify the glycosylphosphatidylinositol (GPI)-anchored peptides in 19 human cancer cell lines. GPI-anchored proteins (GPI-APs), which localize to the outer leaflet of the membrane microdomains commonly referred to as lipid rafts play important roles in diverse biological processes. Due to the complex structure of the GPI-anchor moiety, it has been difficult to identify GPI-anchored peptide sequences on the proteomic scale by database searches using tools such as MASCOT. Here we provide data from 73 ω-sites derived from 49 GPI-APs in 19 human cancer cell lines. This article contains data related to the research article entitled “Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment” (Masuishi et al., 2016) [1]. PMID:27141528

  2. Data for identification of GPI-anchored peptides and ω-sites in cancer cell lines.

    PubMed

    Masuishi, Yusuke; Kimura, Yayoi; Arakawa, Noriaki; Hirano, Hisashi

    2016-06-01

    We present data obtained using a focused proteomics approach to identify the glycosylphosphatidylinositol (GPI)-anchored peptides in 19 human cancer cell lines. GPI-anchored proteins (GPI-APs), which localize to the outer leaflet of the membrane microdomains commonly referred to as lipid rafts play important roles in diverse biological processes. Due to the complex structure of the GPI-anchor moiety, it has been difficult to identify GPI-anchored peptide sequences on the proteomic scale by database searches using tools such as MASCOT. Here we provide data from 73 ω-sites derived from 49 GPI-APs in 19 human cancer cell lines. This article contains data related to the research article entitled "Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment" (Masuishi et al., 2016) [1]. PMID:27141528

  3. A Mixed-Integer Optimization Framework for De Novo Peptide Identification

    PubMed Central

    DiMaggio, Peter A.

    2009-01-01

    A novel methodology for the de novo identification of peptides by mixed-integer optimization and tandem mass spectrometry is presented in this article. The various features of the mathematical model are presented and examples are used to illustrate the key concepts of the proposed approach. Several problems are examined to illustrate the proposed method's ability to address (1) residue-dependent fragmentation properties and (2) the variability of resolution in different mass analyzers. A preprocessing algorithm is used to identify important m/z values in the tandem mass spectrum. Missing peaks, resulting from residue-dependent fragmentation characteristics, are dealt with using a two-stage algorithmic framework. A cross-correlation approach is used to resolve missing amino acid assignments and to identify the most probable peptide by comparing the theoretical spectra of the candidate sequences that were generated from the MILP sequencing stages with the experimental tandem mass spectrum. PMID:19412358

  4. Molecular identification and characterization of peptide: N-glycanase from Schizosaccharomyces pombe

    SciTech Connect

    Xin Fengxue; Wang Shengjun; Song Lei; Liang Quanfeng; Qi Qingsheng

    2008-04-18

    Peptide:N-glycanase (PNGase) is an enzyme responsible for deglycosylation of misfolded glycoproteins in so-called endoplasmic reticulum-associated degradation (ERAD) system. In this study, we reported the molecular identification and characterization of SpPNGase (Schizosaccharomyces pombe PNGase). Enzymatic analysis revealed that SpPNGase deglycosylated the misfolded glycoproteins and distinguished native and denatured high-mannose glycoproteins in vitro. The deglycosylation activity was lost with the addition of chelating agent EDTA and was not restored by re-addition of metal ions. By construction of deletion mutant, we confirmed that N-terminal {alpha}-helix of SpPNGase was responsible for the protein-protein interaction. Combining the results from ternary structure prediction and dendrogram analysis, we suggested that the N-terminal {alpha}-helices of PNGase are derived from evolutionary motif/peptide fusion.

  5. High-sensitivity HLA class I peptidome analysis enables a precise definition of peptide motifs and the identification of peptides from cell lines and patients' sera.

    PubMed

    Ritz, Danilo; Gloger, Andreas; Weide, Benjamin; Garbe, Claus; Neri, Dario; Fugmann, Tim

    2016-05-01

    The characterization of peptides bound to human leukocyte antigen (HLA) class I is of fundamental importance for understanding CD8+ T cell-driven immunological processes and for the development of immunomodulatory therapeutic strategies. However, until now, the mass spectrometric analysis of HLA-bound peptides has typically required billions of cells, still resulting in relatively few high-confidence peptide identifications. Capitalizing on the recent developments in mass spectrometry and bioinformatics, we have implemented a methodology for the efficient recovery of acid-eluted HLA peptides after purification with the pan-reactive antibody W6/32 and have identified a total of 27 862 unique peptides with high confidence (1% false discovery rate) from five human cancer cell lines. More than 93% of the identified peptides were eight to 11 amino acids in length and contained signatures that were in excellent agreement with published HLA binding motifs. Furthermore, by purifying soluble HLA class I complexes (sHLA) from sera of melanoma patients, up to 972 high-confidence peptides could be identified, including melanoma-associated antigens already described in the literature. Knowledge of the HLA class I peptidome should facilitate multiplex tetramer technology-based characterization of T cells, and allow the development of patient selection, stratification and immunomodulatory therapeutic strategies. PMID:26992070

  6. Neuropeptide F peptides act through unique signaling pathways to affect cardiac activity.

    PubMed

    Setzu, M; Biolchini, M; Lilliu, A; Manca, M; Muroni, P; Poddighe, S; Bass, C; Angioy, A M; Nichols, R

    2012-02-01

    Elucidating how neuropeptides affect physiology may result in delineating peptidergic mechanisms and identifying antagonists for application in basic and translational science. Human neuropeptide Y (NPY) regulates cardiac activity; frequently invertebrates contain orthologs of vertebrate peptides. We report invertebrate NPY-like neuropeptide F (NPF) arrested the signal frequency of the slow phase of the cardiac cycle (EC50 = 1 pM); however, signal frequency of the fast phase was affected only minimally. Neuropeptide F decreased the duration of the slow phase by ~70% (EC50 = 0.6 pM), but increased the duration of the fast phase by ~57% (EC50 = 10nM). Short NPF-1 (sNPF-1) decreased the signal frequency of the slow phase by ~70% (EC50 = 9 nM); yet, signal frequency of the fast phase was unaffected. Short NPF-1 decreased the duration of the slow phase ~55% (EC50 ~50 nM), but increased the duration of the fast phase ~20% without dose dependency. Neuropeptide F and sNPF-1 increased isoelectric period duration. This novel report demonstrated NPY-like peptides are cardioactive but functionally unique. These data contribute to understanding how invertebrate orthologs affect cardiovascular activity. Dipteran fast and slow phases may be generated from separate pacemakers in the abdominal heart and in the anterior thoracocephalic aorta, respectively. Thus, our research suggests NPF and sNPF-1 act through different mechanisms to regulate cardiac activity. Invertebrate NPY-like peptides act in olfaction and feeding yet mechanisms which are associated with their cardioactive effects remain unknown; our work may provide evidence linking their roles in sensory response and cardiac activity.

  7. Neuropeptide F peptides act through unique signaling pathways to affect cardiac activity

    PubMed Central

    Setzu, M.; Biolchini, M.; Lilliu, A.; Manca, M.; Muroni, P.; Poddighe, S.; Bass, C.; Angioy, A.M.; Nichols, R.

    2012-01-01

    Elucidating how neuropeptides affect physiology may result in delineating peptidergic mechanisms and identifying antagonists for application in basic and translational science. Human neuropeptide Y (NPY) regulates cardiac activity; frequently invertebrates contain orthologs of vertebrate peptides. We report invertebrate NPY-like neuropeptide F (NPF) arrested the signal frequency of the slow phase of the cardiac cycle (EC50 = 1 pM); however, signal frequency of the fast phase was affected only minimally. Neuropeptide F decreased the duration of the slow phase by ~70% (EC50 = 0.6 pM), but increased the duration of the fast phase by ~57% (EC50 = 10 nM). Short NPF-1 (sNPF-1) decreased the signal frequency of the slow phase by ~70% (EC50 = 9 nM); yet, signal frequency of the fast phase was unaffected. Short NPF-1 decreased the duration of the slow phase ~55% (EC50 ~ 50 nM), but increased the duration of the fast phase ~20% without dose dependency. Neuropeptide F and sNPF-1 increased isoelectric period duration. This novel report demonstrated NPY-like peptides are cardioactive but functionally unique. These data contribute to understanding how invertebrate orthologs affect cardiovascular activity. Dipteran fast and slow phases may be generated from separate pacemakers in the abdominal heart and in the anterior thoracocephalic aorta, respectively. Thus, our research suggests NPF and sNPF-1 act through different mechanisms to regulate cardiac activity. Invertebrate NPY-like peptides act in olfaction and feeding yet mechanisms which are associated with their cardioactive effects remain unknown; our work may provide evidence linking their roles in sensory response and cardiac activity. PMID:22289500

  8. Bacterial formyl peptides affect the innate cellular antimicrobial responses of larval Galleria mellonella (Insecta: Lepidoptera).

    PubMed

    Alavo, Thiery B C; Dunphy, Gary B

    2004-04-01

    The non-self cellular (hemocytic) responses of Galleria mellonella larvae, including the attachment to slides and the removal of the bacteria Xenorhabdus nematophila and Bacillus subtilis from the hemolymph, were affected by N-formyl peptides. Both N-formyl methionyl-leucyl-phenylalanine (fMLF) and the ester derivative decreased hemocyte adhesion in vitro, and both elevated hemocyte counts and suppressed the removal of both X. nematophila and B. subtilis from the hemolymph in vivo. The amide derivative and the antagonist tertiary-butoxy-carbonyl-methionyl-leucyl-phenylalanine (tBOC) increased hemocyte attachment to glass. The fMLF suppressed protein discharge from monolayers of granular cells with and without bacterial stimulation, while tBOC stimulated protein discharge. The peptide tBOC offset the effects of fMLF in vitro and in vivo. This is the first report implying the existence of formyl peptide receptors on insect hemocytes in which the compounds fMLF and tBOC inhibited and activated hemocyte activity, respectively.

  9. Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis

    PubMed Central

    Gerdol, Marco; Puillandre, Nicolas; Moro, Gianluca De; Guarnaccia, Corrado; Lucafò, Marianna; Benincasa, Monica; Zlatev, Ventislav; Manfrin, Chiara; Torboli, Valentina; Giulianini, Piero Giulio; Sava, Gianni; Venier, Paola; Pallavicini, Alberto

    2015-01-01

    We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling. PMID:26201648

  10. Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis.

    PubMed

    Gerdol, Marco; Puillandre, Nicolas; De Moro, Gianluca; Guarnaccia, Corrado; Lucafò, Marianna; Benincasa, Monica; Zlatev, Ventislav; Manfrin, Chiara; Torboli, Valentina; Giulianini, Piero Giulio; Sava, Gianni; Venier, Paola; Pallavicini, Alberto

    2015-08-01

    We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.

  11. The attack of the phytopathogens and the trumpet solo: Identification of a novel plant antifungal peptide with distinct fold and disulfide bond pattern.

    PubMed

    Mandal, Santi M; Porto, William F; Dey, Prabuddha; Maiti, Mrinal K; Ghosh, Ananta K; Franco, Octavio L

    2013-10-01

    Phytopathogens cause economic losses in agribusiness. Plant-derived compounds have been proposed to overcome this problem, including the antimicrobial peptides (AMPs). This paper reports the identification of Ps-AFP1, a novel AMP isolated from the Pisum sativum radicle. Ps-AFP1 was purified and evaluated against phytopathogenic fungi, showing clear effectiveness. In silico analyses were performed, suggesting an unusual fold and disulfide bond pattern. A novel fold and a novel AMP class were here proposed, the αβ-trumpet fold and αβ-trumpet peptides, respectively. The name αβ-trumpet was created due to the peptide's fold, which resembles the musical instrument. The Ps-AFP1 mechanism of action was also proposed. Microscopic analyses revealed that Ps-AFP1 could affect the fungus during the hyphal elongation from spore germination. Furthermore, confocal microscopy performed with Ps-AFP1 labeled with FITC shows that the peptide was localized at high concentration along the fungal cell surface. Due to low cellular disruption rates, it seems that the main target is the fungal cell wall. The binding thermogram and isothermal titration, molecular dynamics and docking analyses were also performed, showing that Ps-AFP1 could bind to chitin producing a stable complex. Data here reported provided novel structural-functional insights into the αβ-trumpet peptide fold.

  12. Isolation and structural determination of three peptides from the insect Locusta migratoria. Identification of a deoxyhexose-linked peptide.

    PubMed

    Nakakura, N; Hietter, H; Van Dorsselaer, A; Luu, B

    1992-02-15

    We have isolated three novel peptides from the aqueous extract of the pars intercerebralis of male and female adults of the insect Locusta migratoria. After extensive HPLC purification, the peptides were characterised by automated Edman degradation and electrospray mass spectrometry: one is a 35-residue peptide (3752.3 +/- 1.1 Da) containing six cysteines involved in three intramolecular disulfide bridges, the second is a 36-residue peptide (3919.2 +/- 0.9 Da), also cross-linked by three intramolecular disulfide bridges. This second peptide is post-translationally modified by a single fucose moiety, which was identified by gas chromatography coupled to mass spectrometry. These two peptides show a strong sequence similarity. Additionally, they were also found in larger amounts in the fat body of Locusta; this finding raises the question of their exact site of synthesis. The third peptide (5776.3 +/- 0.9 Da), a 54-residue peptide cross-linked by six intramolecular disulfide bridges, is structurally related to the two other peptides, but to a lesser extent. Mass spectrometry has shown that all the cysteines within these three peptides are involved in intramolecular disulfide bridges; however, the location of these bridges is not yet established and is currently being investigated. A computer search of sequence data banks did not reveal any significant similarity of these three peptides with other known proteins. PMID:1740125

  13. Milk peptides increase iron dialyzability in water but do not affect DMT-1 expression in Caco-2 cells.

    PubMed

    Argyri, Konstantina; Tako, Elad; Miller, Dennis D; Glahn, Raymond P; Komaitis, Michael; Kapsokefalou, Maria

    2009-02-25

    In vitro digestion of milk produces peptide fractions that enhance iron uptake by Caco-2 cells. The objectives of this study were to investigate whether these fractions (a) exert their effect by increasing relative gene expression of DMT-1 in Caco-2 cells and (b) enhance iron dialyzability when added in meals. Two milk peptide fractions that solubilize iron were isolated by Sephadex G-25 gel filtration of a milk digest. These peptide fractions did not affect relative gene expression of DMT-1 when incubated with Caco-2 cells for 2 or 48 h. Dialyzability was measured after in vitro simulated gastric and pancreatic digestion. Both peptide fractions enhanced the dialyzability of iron from ferric chloride added to PIPES buffer, but had no effect on dialyzability from milk or a vegetable or fruit meal after in vitro simulated gastric and pancreatic digestion. However, dialyzability from milk was enhanced by the addition of a more concentrated lyophilized peptide fraction.

  14. Reaction of phosphorylated and O-glycosylated peptides by chemically targeted identification at ambient temperature.

    PubMed

    Rusnak, Felicia; Zhou, Jie; Hathaway, Gary M

    2004-12-01

    Conditions for carrying out chemically targeted identification of peptides containing phosphorylated or glycosylated serine residues have been investigated. Ba(OH)2 was used at ambient temperature to catalyze the beta-elimination reaction at 25 degrees C. Nucleophilic addition of 2-aminoethanethiol was performed in both parallel and tandem experiments. The method was demonstrated by the reaction of beta-casein tryptic digest phosphopeptides and an O-glycosylated peptide. Contrary to an earlier report by others, the glycopeptide was found to react with essentially the same kinetics as phosphopeptides. Conversion of four phosphoserines in residues 15, 17, 18, and 19 from bovine beta-casein N-terminal tryptic phosphopeptides were followed by monitoring the time course of the addition reaction. The chemistry proceeded rapidly at room temperature with a half-reaction time of 15 min. No side-reaction products were observed; however, care was taken to minimize all counter ions that either precipitate barium or neutralize the base. Digestion of the converted peptides with lysine endopeptidase identified all five phosphoserines in the beta-casein tryptic digest. Alternatively, preincubation with base followed by nucleophilic addition of the thiol was found to work satisfactorily. The use of the water-soluble hydrochloride of 2-aminoethanethiol allowed beta-elimination, nucleophilic addition, and desalting to be carried out on a micro C18 reverse phase pipette tip. PMID:15585826

  15. The Effect of Peptide Identification Search Algorithms on MS2-Based Label-Free Protein Quantification

    PubMed Central

    Degroeve, Sven; Staes, An; De Bock, Pieter-Jan

    2012-01-01

    Abstract Several approaches exist for the quantification of proteins in complex samples processed by liquid chromatography-mass spectrometry followed by fragmentation analysis (MS2). One of these approaches is label-free MS2-based quantification, which takes advantage of the information computed from MS2 spectrum observations to estimate the abundance of a protein in a sample. As a first step in this approach, fragmentation spectra are typically matched to the peptides that generated them by a search algorithm. Because different search algorithms identify overlapping but non-identical sets of peptides, here we investigate whether these differences in peptide identification have an impact on the quantification of the proteins in the sample. We therefore evaluated the effect of using different search algorithms by examining the reproducibility of protein quantification in technical repeat measurements of the same sample. From our results, it is clear that a search engine effect does exist for MS2-based label-free protein quantification methods. As a general conclusion, it is recommended to address the overall possibility of search engine-induced bias in the protein quantification results of label-free MS2-based methods by performing the analysis with two or more distinct search engines. PMID:22804230

  16. Identification and characterization of antimicrobial peptides from skin of Amolops ricketti (Anura: Ranidae).

    PubMed

    Wang, Hui; Ran, Ran; Yu, Haining; Yu, Zhijun; Hu, Yuhong; Zheng, Hongyuan; Wang, Duo; Yang, Fan; Liu, Renjie; Liu, Jingze

    2012-01-01

    As one of large amphibian group, there are a total of 45 species of Amolops in the world. However, the antimicrobial peptides (AMPs) existing in this genus has not been extensively studied. In this study, cDNAs encoding five novel AMP precursors were cloned by screening the skin-derived cDNA library of Amolops ricketti, a frog species that exists in southern and western parts of China. Protein sequence analysis led to the identification of five deduced peptides, three belonging to the brevinin-1 family and two belonging to the brevinin-2 family of amphibian AMPs. Thus, they were named as brevinin-1RTa (FLPLLAGVVANFLPQIICKIARKC), brevinin-1RTb (FLGSLLGLVGKVVPTLFCKISKKC), brevinin-1RTc (FLGSLLGLVGKIVPTLICKISKKC), brevinin-2RTa (GLMSTLKDFGKTAAKEIAQSLLSTASCKLAKTC), and brevinin-2RTb (GILDTLKEFGKTAAKGIAQSLLSTASCKLAKTC), respectively. The purification of brevinin-1RTa, brevinin-1RTb, and brevinin-2RTb was carried out by RP-HPLC, and confirmed by the LC-MS/MS-based proteomics approach. All of the peptides displayed different antimicrobial potency against a variety of microorganisms. In addition, brevinin-2RTa and brevinin-2RTb were found to have relatively low hemolytic activity (>400μg/ml) against mammalian red blood cells in vitro, which could potentially be as candidates for developing novel anti-infection agents.

  17. Spectrum-based method to generate good decoy libraries for spectral library searching in peptide identifications.

    PubMed

    Cheng, Chia-Ying; Tsai, Chia-Feng; Chen, Yu-Ju; Sung, Ting-Yi; Hsu, Wen-Lian

    2013-05-01

    As spectral library searching has received increasing attention for peptide identification, constructing good decoy spectra from the target spectra is the key to correctly estimating the false discovery rate in searching against the concatenated target-decoy spectral library. Several methods have been proposed to construct decoy spectral libraries. Most of them construct decoy peptide sequences and then generate theoretical spectra accordingly. In this paper, we propose a method, called precursor-swap, which directly constructs decoy spectral libraries directly at the "spectrum level" without generating decoy peptide sequences by swapping the precursors of two spectra selected according to a very simple rule. Our spectrum-based method does not require additional efforts to deal with ion types (e.g., a, b or c ions), fragment mechanism (e.g., CID, or ETD), or unannotated peaks, but preserves many spectral properties. The precursor-swap method is evaluated on different spectral libraries and the results of obtained decoy ratios show that it is comparable to other methods. Notably, it is efficient in time and memory usage for constructing decoy libraries. A software tool called Precursor-Swap-Decoy-Generation (PSDG) is publicly available for download at http://ms.iis.sinica.edu.tw/PSDG/.

  18. Maximizing the sensitivity and reliability of peptide identification in large-scale proteomic experiments by harnessing multiple search engines.

    PubMed

    Yu, Wen; Taylor, J Alex; Davis, Michael T; Bonilla, Leo E; Lee, Kimberly A; Auger, Paul L; Farnsworth, Chris C; Welcher, Andrew A; Patterson, Scott D

    2010-03-01

    Despite recent advances in qualitative proteomics, the automatic identification of peptides with optimal sensitivity and accuracy remains a difficult goal. To address this deficiency, a novel algorithm, Multiple Search Engines, Normalization and Consensus is described. The method employs six search engines and a re-scoring engine to search MS/MS spectra against protein and decoy sequences. After the peptide hits from each engine are normalized to error rates estimated from the decoy hits, peptide assignments are then deduced using a minimum consensus model. These assignments are produced in a series of progressively relaxed false-discovery rates, thus enabling a comprehensive interpretation of the data set. Additionally, the estimated false-discovery rate was found to have good concordance with the observed false-positive rate calculated from known identities. Benchmarking against standard proteins data sets (ISBv1, sPRG2006) and their published analysis, demonstrated that the Multiple Search Engines, Normalization and Consensus algorithm consistently achieved significantly higher sensitivity in peptide identifications, which led to increased or more robust protein identifications in all data sets compared with prior methods. The sensitivity and the false-positive rate of peptide identification exhibit an inverse-proportional and linear relationship with the number of participating search engines.

  19. Peptide neurotoxins that affect voltage-gated calcium channels: a close-up on ω-agatoxins.

    PubMed

    Pringos, Emilie; Vignes, Michel; Martinez, Jean; Rolland, Valerie

    2011-01-01

    Peptide neurotoxins found in animal venoms have gained great interest in the field of neurotransmission. As they are high affinity ligands for calcium, potassium and sodium channels, they have become useful tools for studying channel structure and activity. Peptide neurotoxins represent the clinical potential of ion-channel modulators across several therapeutic fields, especially in developing new strategies for treatment of ion channel-related diseases. The aim of this review is to overview the latest updates in the domain of peptide neurotoxins that affect voltage-gated calcium channels, with a special focus on ω-agatoxins.

  20. Nearest Neighbor Interactions Affect the Conformational Distribution in the Unfolded State of Peptides

    NASA Astrophysics Data System (ADS)

    Toal, Siobhan; Schweitzer-Stenner, Reinhard; Rybka, Karin; Schwalbe, Hardol

    2013-03-01

    In order to enable structural predictions of intrinsically disordered proteins (IDPs) the intrinsic conformational propensities of amino acids must be complimented by information on nearest-neighbor interactions. To explore the influence of nearest-neighbors on conformational distributions, we preformed a joint vibrational (Infrared, Vibrational Circular Dichroism (VCD), polarized Raman) and 2D-NMR study of selected GxyG host-guest peptides: GDyG, GSyG, GxLG, GxVG, where x/y ={A,K,LV}. D and S (L and V) were chosen at the x (y) position due to their observance to drastically change the distribution of alanine in xAy tripeptide sequences in truncated coil libraries. The conformationally sensitive amide' profiles of the respective spectra were analyzed in terms of a statistical ensemble described as a superposition of 2D-Gaussian functions in Ramachandran space representing sub-ensembles of pPII-, β-strand-, helical-, and turn-like conformations. Our analysis and simulation of the amide I' band profiles exploits excitonic coupling between the local amide I' vibrational modes in the tetra-peptides. The resulting distributions reveal that D and S, which themselves have high propensities for turn-structures, strongly affect the conformational distribution of their downstream neighbor. Taken together, our results indicate that Dx and Sx motifs might act as conformational randomizers in proteins, attenuating intrinsic propensities of neighboring residues. Overall, our results show that nearest neighbor interactions contribute significantly to the Gibbs energy landscape of disordered peptides and proteins.

  1. Employees' organizational identification and affective organizational commitment: an integrative approach.

    PubMed

    Stinglhamber, Florence; Marique, Géraldine; Caesens, Gaëtane; Desmette, Donatienne; Hansez, Isabelle; Hanin, Dorothée; Bertrand, Françoise

    2015-01-01

    Although several studies have empirically supported the distinction between organizational identification (OI) and affective commitment (AC), there is still disagreement regarding how they are related. Precisely, little attention has been given to the direction of causality between these two constructs and as to why they have common antecedents and outcomes. This research was designed to fill these gaps. Using a cross-lagged panel design with two measurement times, Study 1 examined the directionality of the relationship between OI and AC, and showed that OI is positively related to temporal change in AC, confirming the antecedence of OI on AC. Using a cross-sectional design, Study 2 investigated the mediating role of OI in the relationship between three work experiences (i.e., perceived organizational support, leader-member exchange, and job autonomy) and AC, and found that OI partially mediates the influence of work experiences on AC. Finally, Study 3 examined longitudinally how OI and AC combine in the prediction of actual turnover, and showed that AC totally mediates the relationship between OI and turnover. Overall, these findings suggest that favorable work experiences operate via OI to increase employees' AC that, in turn, decreases employee turnover.

  2. Employees’ Organizational Identification and Affective Organizational Commitment: An Integrative Approach

    PubMed Central

    Stinglhamber, Florence; Marique, Géraldine; Caesens, Gaëtane; Desmette, Donatienne; Hansez, Isabelle; Hanin, Dorothée; Bertrand, Françoise

    2015-01-01

    Although several studies have empirically supported the distinction between organizational identification (OI) and affective commitment (AC), there is still disagreement regarding how they are related. Precisely, little attention has been given to the direction of causality between these two constructs and as to why they have common antecedents and outcomes. This research was designed to fill these gaps. Using a cross-lagged panel design with two measurement times, Study 1 examined the directionality of the relationship between OI and AC, and showed that OI is positively related to temporal change in AC, confirming the antecedence of OI on AC. Using a cross-sectional design, Study 2 investigated the mediating role of OI in the relationship between three work experiences (i.e., perceived organizational support, leader-member exchange, and job autonomy) and AC, and found that OI partially mediates the influence of work experiences on AC. Finally, Study 3 examined longitudinally how OI and AC combine in the prediction of actual turnover, and showed that AC totally mediates the relationship between OI and turnover. Overall, these findings suggest that favorable work experiences operate via OI to increase employees' AC that, in turn, decreases employee turnover. PMID:25875086

  3. Employees' organizational identification and affective organizational commitment: an integrative approach.

    PubMed

    Stinglhamber, Florence; Marique, Géraldine; Caesens, Gaëtane; Desmette, Donatienne; Hansez, Isabelle; Hanin, Dorothée; Bertrand, Françoise

    2015-01-01

    Although several studies have empirically supported the distinction between organizational identification (OI) and affective commitment (AC), there is still disagreement regarding how they are related. Precisely, little attention has been given to the direction of causality between these two constructs and as to why they have common antecedents and outcomes. This research was designed to fill these gaps. Using a cross-lagged panel design with two measurement times, Study 1 examined the directionality of the relationship between OI and AC, and showed that OI is positively related to temporal change in AC, confirming the antecedence of OI on AC. Using a cross-sectional design, Study 2 investigated the mediating role of OI in the relationship between three work experiences (i.e., perceived organizational support, leader-member exchange, and job autonomy) and AC, and found that OI partially mediates the influence of work experiences on AC. Finally, Study 3 examined longitudinally how OI and AC combine in the prediction of actual turnover, and showed that AC totally mediates the relationship between OI and turnover. Overall, these findings suggest that favorable work experiences operate via OI to increase employees' AC that, in turn, decreases employee turnover. PMID:25875086

  4. Identification of a novel lytic peptide for the treatment of solid tumours

    PubMed Central

    Szczepanski, Claudia; Tenstad, Olav; Baumann, Anne; Martinez, Aurora; Myklebust, Reidar

    2014-01-01

    Originally known as host defence peptides for their substantial bacteriotoxic effects, many cationic antimicrobial peptides also exhibit a potent cytotoxic activity against cancer cells. Their mode of action is characterized mostly by electrostatic interactions with the plasma membrane, leading to membrane disruption and rapid necrotic cell death. In this work, we have designed a novel cationic peptide of 27 amino acids (Cypep-1), which shows efficacy against a number of cancer cell types, both in vitro and in vivo, while normal human fibroblasts were significantly less affected. Surface plasmon resonance experiments as well as liposome leakage assays monitored by fluorescence spectroscopy revealed a substantial binding affinity of Cypep-1 to negatively charged liposomes and induced significant leakage of liposome content after exposure to the peptide. The observed membranolytic effect of Cypep-1 was confirmed by scanning electron microscopy (SEM) as well as by time-lapse confocal microscopy. Pharmacokinetic profiling of Cypep-1 in rats showed a short plasma half-life after i.v. injection, followed mainly by retention in the liver, spleen and kidneys. Extremely low concentrations within the organs of the central nervous system indicated that Cypep-1 did not pass the blood-brain-barrier. Local treatment of 4T1 murine mammary carcinoma allografts by means of a single local bolus injection of Cypep-1 led to a significant reduction of tumour growth in the following weeks and prolonged survival. Detailed histological analysis of the treated tumours revealed large areas of necrosis. In sum, our findings show that the novel cationic peptide Cypep-1 displays a strong cytolytic activity against cancer cells both in vitro and in vivo and thus holds a substantial therapeutic potential. PMID:25061502

  5. Identification of lactoferrin peptides generated by digestion with human gastrointestinal enzymes.

    PubMed

    Furlund, C B; Ulleberg, E K; Devold, T G; Flengsrud, R; Jacobsen, M; Sekse, C; Holm, H; Vegarud, G E

    2013-01-01

    Lactoferrin (LF) is a protein present in milk and other body fluids that plays important biological roles. As part of a diet, LF must survive gastrointestinal conditions or create bioactive fragments to exert its effects. The degradation of LF and formation of bioactive peptides is highly dependent on individual variation in intraluminal composition. The present study was designed to compare the degradation and peptide formation of bovine LF (bLF) following in vitro digestion under different simulated intraluminal conditions. Human gastrointestinal (GI) juices were used in a 2-step model digestion to mimic degradation in the stomach and duodenum. To account for variation in the buffering capacity of different lactoferrin-containing foods, gastric pH was adjusted either slowly or rapidly to 2.5 or 4.0. The results were compared with in vivo digestion of bLF performed in 2 volunteers. High concentration of GI juices and fast pH reduction to 2.5 resulted in complete degradation in the gastric step. More LF resisted gastric digestion when pH was slowly reduced to 2.5 or 4.0. Several peptides were identified; however, few matched with previously reported peptides from studies using nonhuman enzymes. Surprisingly, no bovine lactoferricin, f(17-41), was identified during in vitro or in vivo digestion under the intraluminal conditions used. The diversity of enzymes in human GI juices seems to affect the hydrolysis of bLF, generating different peptide fragments compared with those obtained when using only one or a few proteases of animal origin. Multiple sequence analysis of the identified peptides indicated a motif consisting of proline and neighboring hydrophobic residues that could restrict proteolytic processing. Further structure analysis showed that almost all proteolytic cutting sites were located on the surface and mainly on the nonglycosylated half of lactoferrin. Digestion of bLF by human enzymes may generate different peptides from those found when lactoferrin is

  6. Identification of bioactive peptides in hypoallergenic infant milk formulas by capillary electrophoresis-mass spectrometry.

    PubMed

    Català-Clariana, Sergio; Benavente, Fernando; Giménez, Estela; Barbosa, José; Sanz-Nebot, Víctoria

    2010-12-17

    In this study, we use capillary electrophoresis-mass spectrometry (CE-MS) for the identification of bioactive peptides in hypoallergenic infant milk formulas (IF), which are complex bovine milk protein hydrolysates. A sample clean-up pretreatment with a citrate buffer containing dithiothreitol and urea followed by solid-phase extraction (SPE) with different reversed-phase commercial cartridges was investigated to achieve optimum detection sensitivity in CE-MS. SPE with C18, StrataX and Oasis HLB cartridges allowed detection of the largest number of low molecular mass components, but combination of C18 and StrataX results was enough to achieve an excellent coverage of the studied IF. The monoisotopic molecular mass values of the low molecular mass components obtained by capillary electrophoresis ion-trap mass spectrometry (CE-IT-MS) allowed the tentative identification of nine bioactive sequences. Only the identification of five of them could be confirmed when accurate mass measurements were performed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS), namely LKP, IPY, ALPM, PGPIHN and VAGTWY, which were reported to present angiotensin-converting enzyme (ACE) inhibitory and antimicrobial activity (only VAGTWY).

  7. Accelerating the scoring module of mass spectrometry-based peptide identification using GPUs

    PubMed Central

    2014-01-01

    Background Tandem mass spectrometry-based database searching is currently the main method for protein identification in shotgun proteomics. The explosive growth of protein and peptide databases, which is a result of genome translations, enzymatic digestions, and post-translational modifications (PTMs), is making computational efficiency in database searching a serious challenge. Profile analysis shows that most search engines spend 50%-90% of their total time on the scoring module, and that the spectrum dot product (SDP) based scoring module is the most widely used. As a general purpose and high performance parallel hardware, graphics processing units (GPUs) are promising platforms for speeding up database searches in the protein identification process. Results We designed and implemented a parallel SDP-based scoring module on GPUs that exploits the efficient use of GPU registers, constant memory and shared memory. Compared with the CPU-based version, we achieved a 30 to 60 times speedup using a single GPU. We also implemented our algorithm on a GPU cluster and achieved an approximately favorable speedup. Conclusions Our GPU-based SDP algorithm can significantly improve the speed of the scoring module in mass spectrometry-based protein identification. The algorithm can be easily implemented in many database search engines such as X!Tandem, SEQUEST, and pFind. A software tool implementing this algorithm is available at http://www.comp.hkbu.edu.hk/~youli/ProteinByGPU.html PMID:24773593

  8. Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide.

    PubMed

    Nakrieko, Kerry-Ann; Mould, Ruth M; Smith, Alison G

    2004-02-01

    Phosphorylation of the transit peptide of several chloroplast-targeted proteins enables the binding of 14-3-3 proteins. The complex that forms, together with Hsp70, has been demonstrated to be an intermediate in the chloroplast protein import pathway in vitro[May, T. & Soll, J. (2000) Plant Cell 12, 53-63]. In this paper we report that mutagenesis (in order to remove the phosphorylation site) of the transit peptide of the small subunit of ribulose bisphosphate carboxylase/oxygenase did not affect its ability to target green fluorescent protein to chloroplasts in vivo. We also found no mistargeting to other organelles such as mitochondria. Similar alterations to the transit peptides of histidyl- or cysteinyl-tRNA synthetase, which are dual-targeted to chloroplasts and mitochondria, had no effect on their ability to target green fluorescent protein in vivo. Thus, phosphorylation of the transit peptide is not responsible for the specificity of chloroplast import.

  9. Identification of non-random sequence properties in groups of signature peptides obtained in random sequence peptide microarray experiments.

    PubMed

    Kuznetsov, Igor B

    2016-05-01

    Immunosignaturing is an emerging experimental technique that uses random sequence peptide microarrays to detect antibodies produced by the immune system in response to a particular disease. Two important questions regarding immunosignaturing are "Do microarray peptides that exhibit a strong affinity to a given type of antibodies share common sequence properties?" and "If so, what are those properties?" In this work, three statistical tests designed to detect non-random patterns in the amino acid makeup of a group of microarray peptides are presented. One test detects patterns of significantly biased amino acid usage, whereas the other two detect patterns of significant bias in the biochemical properties. These tests do not require a large number of peptides per group. The tests were applied to analyze 19 groups of peptides identified in immunosignaturing experiments as being specific for antibodies produced in response to various types of cancer and other diseases. The positional distribution of the biochemical properties of the amino acids in these 19 peptide groups was also studied. Remarkably, despite the random nature of the sequence libraries used to design the microarrays, a unique group-specific non-random pattern was identified in the majority of the peptide groups studied. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 318-329, 2016. PMID:27037995

  10. Identification and characterization of latency-associated peptide-expressing γδ T cells

    PubMed Central

    Rezende, Rafael M.; da Cunha, Andre P.; Kuhn, Chantal; Rubino, Stephen; M'Hamdi, Hanane; Gabriely, Galina; Vandeventer, Tyler; Liu, Shirong; Cialic, Ron; Pinheiro-Rosa, Natalia; Oliveira, Rafael P.; Gaublomme, Jellert T.; Obholzer, Nikolaus; Kozubek, James; Pochet, Nathalie; Faria, Ana M. C.; Weiner, Howard L.

    2015-01-01

    γδ T cells are a subset of lymphocytes specialized in protecting the host against pathogens and tumours. Here we describe a subset of regulatory γδ T cells that express the latency-associated peptide (LAP), a membrane-bound TGF-β1. Thymic CD27+IFN-γ+CCR9+α4β7+TCRγδ+ cells migrate to the periphery, particularly to Peyer's patches and small intestine lamina propria, where they upregulate LAP, downregulate IFN-γ via ATF-3 expression and acquire a regulatory phenotype. TCRγδ+LAP+ cells express antigen presentation molecules and function as antigen presenting cells that induce CD4+Foxp3+ regulatory T cells, although TCRγδ+LAP+ cells do not themselves express Foxp3. Identification of TCRγδ+LAP+ regulatory cells provides an avenue for understanding immune regulation and biologic processes linked to intestinal function and disease. PMID:26644347

  11. MyriMatch: highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis

    PubMed Central

    Tabb, David L.; Fernando, Christopher G.; Chambers, Matthew C.

    2008-01-01

    Shotgun proteomics experiments are dependent upon database search engines to identify peptides from tandem mass spectra. Many of these algorithms score potential identifications by evaluating the number of fragment ions matched between each peptide sequence and an observed spectrum. These systems, however, generally do not distinguish between matching an intense peak and matching a minor peak. We have developed a statistical model to score peptide matches that is based upon the multivariate hypergeometric distribution. This scorer, part of the “MyriMatch” database search engine, places greater emphasis on matching intense peaks. The probability that the best match for each spectrum has occurred by random chance can be employed to separate correct matches from random ones. We evaluated this software on data sets from three different laboratories employing three different ion trap instruments. Employing a novel system for testing discrimination, we demonstrate that stratifying peaks into multiple intensity classes improves the discrimination of scoring. We compare MyriMatch results to those of Sequest and X!Tandem, revealing that it is capable of higher discrimination than either of these algorithms. When minimal peak filtering is employed, performance plummets for a scoring model that does not stratify matched peaks by intensity. On the other hand, we find that MyriMatch discrimination improves as more peaks are retained in each spectrum. MyriMatch also scales well to tandem mass spectra from high-resolution mass analyzers. These findings may indicate limitations for existing database search scorers that count matched peaks without differentiating them by intensity. This software and source code is available under Mozilla Public License at this URL: http://www.mc.vanderbilt.edu/msrc/bioinformatics/. PMID:17269722

  12. Bayesian Nonparametric Model for the Validation of Peptide Identification in Shotgun Proteomics*S⃞

    PubMed Central

    Zhang, Jiyang; Ma, Jie; Dou, Lei; Wu, Songfeng; Qian, Xiaohong; Xie, Hongwei; Zhu, Yunping; He, Fuchu

    2009-01-01

    Tandem mass spectrometry combined with database searching allows high throughput identification of peptides in shotgun proteomics. However, validating database search results, a problem with a lot of solutions proposed, is still advancing in some aspects, such as the sensitivity, specificity, and generalizability of the validation algorithms. Here a Bayesian nonparametric (BNP) model for the validation of database search results was developed that incorporates several popular techniques in statistical learning, including the compression of feature space with a linear discriminant function, the flexible nonparametric probability density function estimation for the variable probability structure in complex problem, and the Bayesian method to calculate the posterior probability. Importantly the BNP model is compatible with the popular target-decoy database search strategy naturally. We tested the BNP model on standard proteins and real, complex sample data sets from multiple MS platforms and compared it with PeptideProphet, the cutoff-based method, and a simple nonparametric method (proposed by us previously). The performance of the BNP model was shown to be superior for all data sets searched on sensitivity and generalizability. Some high quality matches that had been filtered out by other methods were detected and assigned with high probability by the BNP model. Thus, the BNP model could be able to validate the database search results effectively and extract more information from MS/MS data. PMID:19005226

  13. Neuropeptides in Heteroptera: Identification of allatotropin-related peptide and tachykinin-related peptides using MALDI-TOF mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, the peptidomic analysis of neuropeptides from the retrocerebral complex and abdominal perisympathetic organs of polyphagous stinkbugs (Pentatomidae) revealed the group-specific sequences of pyrokinins, CAPA peptides (CAPA-periviscerokinins/PVKs and CAPA-pyrokinin), myosuppressin, corazonin...

  14. Identification of peptides using gold nanoparticle-assisted single-drop microextraction coupled with AP-MALDI mass spectrometry.

    PubMed

    Sudhir, Putty-Reddy; Wu, Hui-Fen; Zhou, Zi-Cong

    2005-11-15

    A novel technique, gold nanoparticle-assisted single-drop microextraction (SDME) combined with atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) for the identification of peptides has been described. The SDME of peptides from aqueous solution was achieved using gold nanoparticles prepared in toluene as the acceptor phase. A simple phenomenon of isoelectric point (pI) of the peptides has been utilized successfully to extract the peptides into a single drop of nanogold in toluene. After extraction, a single-drop nano gold solution was directly spotted onto the target plate with an equal volume of matrix, proportional, variant-cyanohydroxy cinnamic acid ( proportional, variant-CHCA) and analyzed in AP-MALDI-MS. The parameters, such as solvent selection, extraction time, agitation rate, and pH effect, were optimized for the SDME technique. Using this technique, in aqueous solution, the lowest concentration detected for Met- and Leu-enkephalin peptides was 0.2 and 0.17 microM, respectively. In addition, the application of this technique to obtain the signal for the selected peptides in a mass spectrum in the presence of matrix interferences such as 1% Triton X-100 and 6.5 M urea has been showed. The application was extended to identify the peptides spiked into urine.

  15. Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library

    PubMed Central

    Wu, Yan; Li, Cai-Yun

    2015-01-01

    Objective Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy. Methods The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides. Results After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with Kaff (affinity constant) of 16.4±8.6 µg/mL (n=3), 9.2±2.1 µg/mL (n=3), and 174.8±31.1 µg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed. Conclusion These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer. PMID:26197772

  16. Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides.

    PubMed

    Cascales, Laura; Henriques, Sónia T; Kerr, Markus C; Huang, Yen-Hua; Sweet, Matthew J; Daly, Norelle L; Craik, David J

    2011-10-21

    Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides. PMID:21873420

  17. Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides.

    PubMed

    Cascales, Laura; Henriques, Sónia T; Kerr, Markus C; Huang, Yen-Hua; Sweet, Matthew J; Daly, Norelle L; Craik, David J

    2011-10-21

    Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.

  18. Identification and functional characterization of a novel locust peptide belonging to the family of insect growth blocking peptides.

    PubMed

    Duressa, Tewodros Firdissa; Boonen, Kurt; Hayakawa, Yoichi; Huybrechts, Roger

    2015-12-01

    Growth blocking peptides (GBPs) are recognized as insect cytokines that take part in multifaceted functions including immune system activation and growth retardation. The peptides induce hemocyte spreading in vitro, which is considered as the initial step in hemocyte activation against infection in many insect species. Therefore, in this study, we carried out a series of in vitro bioassay driven fractionations of Locusta migratoria hemolymph combined with mass spectrometry to identify locust hemocyte activation factors belonging to the family of insect GBPs. We identified the locust hemocyte spreading peptide (locust GBP) as a 28-mer peptide encoded at the C-terminus of a 64 amino acid long precursor polypeptide. As demonstrated by QRT-PCR, the gene encoding the locust GBP precursor (proGBP) was expressed in large quantities in diverse locust tissues including fat body, endocrine glands, central nervous system, reproductive tissues and flight muscles. In contrary, hemocytes, gut tissues and Malpighian tubules displayed little expression of the proGBP transcript. The bioactive peptide induces transient depletion of hemocytes in vivo and when injected in last instar nymphs it extends the larval growth phase and postpones adult molting. In addition, we identified a functional homologous hemocyte spreading peptide in Schistocerca gregaria.

  19. Identification of FAK substrate peptides via colorimetric screening of a one-bead one-peptide combinatorial library.

    PubMed

    Witucki, Laurie A; Borowicz, Lauren Sanford; Pedley, Anthony M; Curtis-Fisk, Jaime; Kuszpit, Elizabeth Girnys

    2015-04-01

    Focal adhesion kinase (FAK) is a protein tyrosine kinase that is associated with regulating cellular functions such as cell adhesion and migration and has emerged as an important target for cancer research. Short peptide substrates that are selectively and efficiently phosphorylated by FAK have not been previously identified and tested. Here we report the synthesis and screening of a one-bead one-peptide combinatorial library to identify novel substrates for FAK. Using a solid-phase colorimetric antibody tagging detection platform, the peptide beads phosphorylated by FAK were sequenced via Edman degradation and then validated through radioisotope kinetic studies with [γ-(32)P] ATP to derive Michaelis-Menton constants. The combination of results gathered from both colorimetric and radioisotope kinase assays led to the rational design of a second generation of FAK peptide substrates. Out of all the potential peptide substrates evaluated, the most active was GDYVEFKKK with a K(M)  = 92 μM and a Vmax  = 1920 nmol/min/mg. Peptide substrates discovered within this study may be useful diagnostic tools for future kinase investigations and may lead to novel therapeutic agents.

  20. Identification and functional characterization of a novel locust peptide belonging to the family of insect growth blocking peptides.

    PubMed

    Duressa, Tewodros Firdissa; Boonen, Kurt; Hayakawa, Yoichi; Huybrechts, Roger

    2015-12-01

    Growth blocking peptides (GBPs) are recognized as insect cytokines that take part in multifaceted functions including immune system activation and growth retardation. The peptides induce hemocyte spreading in vitro, which is considered as the initial step in hemocyte activation against infection in many insect species. Therefore, in this study, we carried out a series of in vitro bioassay driven fractionations of Locusta migratoria hemolymph combined with mass spectrometry to identify locust hemocyte activation factors belonging to the family of insect GBPs. We identified the locust hemocyte spreading peptide (locust GBP) as a 28-mer peptide encoded at the C-terminus of a 64 amino acid long precursor polypeptide. As demonstrated by QRT-PCR, the gene encoding the locust GBP precursor (proGBP) was expressed in large quantities in diverse locust tissues including fat body, endocrine glands, central nervous system, reproductive tissues and flight muscles. In contrary, hemocytes, gut tissues and Malpighian tubules displayed little expression of the proGBP transcript. The bioactive peptide induces transient depletion of hemocytes in vivo and when injected in last instar nymphs it extends the larval growth phase and postpones adult molting. In addition, we identified a functional homologous hemocyte spreading peptide in Schistocerca gregaria. PMID:26471907

  1. STEPS: a grid search methodology for optimized peptide identification filtering of MS/MS database search results.

    PubMed

    Piehowski, Paul D; Petyuk, Vladislav A; Sandoval, John D; Burnum, Kristin E; Kiebel, Gary R; Monroe, Matthew E; Anderson, Gordon A; Camp, David G; Smith, Richard D

    2013-03-01

    For bottom-up proteomics, there are wide variety of database-searching algorithms in use for matching peptide sequences to tandem MS spectra. Likewise, there are numerous strategies being employed to produce a confident list of peptide identifications from the different search algorithm outputs. Here we introduce a grid-search approach for determining optimal database filtering criteria in shotgun proteomics data analyses that is easily adaptable to any search. Systematic Trial and Error Parameter Selection--referred to as STEPS--utilizes user-defined parameter ranges to test a wide array of parameter combinations to arrive at an optimal "parameter set" for data filtering, thus maximizing confident identifications. The benefits of this approach in terms of numbers of true-positive identifications are demonstrated using datasets derived from immunoaffinity-depleted blood serum and a bacterial cell lysate, two common proteomics sample types.

  2. STEPS: A Grid Search Methodology for Optimized Peptide Identification Filtering of MS/MS Database Search Results

    SciTech Connect

    Piehowski, Paul D.; Petyuk, Vladislav A.; Sandoval, John D.; Burnum, Kristin E.; Kiebel, Gary R.; Monroe, Matthew E.; Anderson, Gordon A.; Camp, David G.; Smith, Richard D.

    2013-03-01

    For bottom-up proteomics there are a wide variety of database searching algorithms in use for matching peptide sequences to tandem MS spectra. Likewise, there are numerous strategies being employed to produce a confident list of peptide identifications from the different search algorithm outputs. Here we introduce a grid search approach for determining optimal database filtering criteria in shotgun proteomics data analyses that is easily adaptable to any search. Systematic Trial and Error Parameter Selection - referred to as STEPS - utilizes user-defined parameter ranges to test a wide array of parameter combinations to arrive at an optimal "parameter set" for data filtering, thus maximizing confident identifications. The benefits of this approach in terms of numbers of true positive identifications are demonstrated using datasets derived from immunoaffinity-depleted blood serum and a bacterial cell lysate, two common proteomics sample types.

  3. Factors Affecting Isolation and Identification of Haemophilus vaginalis (Corynebacterium vaginale)

    PubMed Central

    Bailey, Robert K.; Voss, Jack L.; Smith, Rodney F.

    1979-01-01

    The rate of isolation of organisms resembling Haemophilus vaginalis (Corynebacterium vaginale) from vaginal specimens was not significantly affected by anaerobic versus carbon dioxide incubation atmospheres or whether specimens were inoculated on isolation media immediately after collection or after a delay of 6 h. Forty-one clinically isolated strains were provisionally divided into 30 H. vaginalis strains and 11 H. vaginalis-like (HVL) strains based on morphological and growth characteristics. The H. vaginalis strains were less reactive in API-20A identification test strips, (Analytab Products, Inc.) using Lombard-Dowell broth, than in a modified basal medium that contained proteose peptone no. 3 (Difco). The numbers and kinds of substrates fermented by 30 clinical and 2 reference strains of H. vaginalis varied among conventional, API, Minitek (Baltimore Biological Laboratory), and rapid buffered substrate fermentation systems. A greater number and variety of carbohydrates were fermented by the 11 HVL strains more consistently in all four test systems. Analysis of volatile and nonvolatile fermentation end products by gas-liquid chromatography did not reveal significant differences between the H. vaginalis and HVL strains. However, the latter group grew in peptone-yeast extract-glucose broth, whereas the H. vaginalis strains did not grow without the addition of starch to peptone-yeast extract-glucose. All of the reference and clinical strains were similar in their susceptibilities to a variety of antimicrobial compounds except sulfonamides, which inhibited the HVL strains and bifidobacteria but not the H. vaginalis strains. Sulfonamide susceptibility or resistance corresponded in part to the H. vaginalis and HVL-bifidobacteria strain reactions on selected conventional fermentation substrates. Susceptibility or resistance to sulfonamides and metronidazole in conjunction with fermentation tests is described to aid in the separation of H. vaginalis from other

  4. Factors affecting isolation and identification of Haemophilus vaginalis (Corynebacterium vaginale).

    PubMed

    Bailey, R K; Voss, J L; Smith, R F

    1979-01-01

    The rate of isolation of organisms resembling Haemophilus vaginalis (Corynebacterium vaginale) from vaginal specimens was not significantly affected by anaerobic versus carbon dioxide incubation atmospheres or whether specimens were inoculated on isolation media immediately after collection or after a delay of 6 h. Forty-one clinically isolated strains were provisionally divided into 30 H. vaginalis strains and 11 H. vaginalis-like (HVL) strains based on morphological and growth characteristics. The H. vaginalis strains were less reactive in API-20A identification test strips, (Analytab Products, Inc.) using Lombard-Dowell broth, than in a modified basal medium that contained proteose peptone no. 3 (Difco). The numbers and kinds of substrates fermented by 30 clinical and 2 reference strains of H. vaginalis varied among conventional, API, Minitek (Baltimore Biological Laboratory), and rapid buffered substrate fermentation systems. A greater number and variety of carbohydrates were fermented by the 11 HVL strains more consistently in all four test systems. Analysis of volatile and nonvolatile fermentation end products by gas-liquid chromatography did not reveal significant differences between the H. vaginalis and HVL strains. However, the latter group grew in peptone-yeast extract-glucose broth, whereas the H. vaginalis strains did not grow without the addition of starch to peptone-yeast extract-glucose. All of the reference and clinical strains were similar in their susceptibilities to a variety of antimicrobial compounds except sulfonamides, which inhibited the HVL strains and bifidobacteria but not the H. vaginalis strains. Sulfonamide susceptibility or resistance corresponded in part to the H. vaginalis and HVL-bifidobacteria strain reactions on selected conventional fermentation substrates. Susceptibility or resistance to sulfonamides and metronidazole in conjunction with fermentation tests is described to aid in the separation of H. vaginalis from other

  5. Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library.

    PubMed

    Choi, Ji Young; Lee, Sang Hyoung; Park, Chan Young; Heo, Won Do; Kim, Jong Cheol; Kim, Min Chul; Chung, Woo Sik; Moon, Byeong Cheol; Cheong, Yong Hwa; Kim, Cha Young; Yoo, Jae Hyuk; Koo, Ja Choon; Ok, Hyun Mi; Chi, Seung-Wook; Ryu, Seong-Eon; Lee, Sang Yeol; Lim, Chae Oh; Cho, Moo Je

    2002-06-14

    Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.

  6. Factors affecting the identification of individual mountain bongo antelope.

    PubMed

    Gibbon, Gwili E M; Bindemann, Markus; Roberts, David L

    2015-01-01

    The recognition of individuals forms the basis of many endangered species monitoring protocols. This process typically relies on manual recognition techniques. This study aimed to calculate a measure of the error rates inherent within the manual technique and also sought to identify visual traits that aid identification, using the critically endangered mountain bongo, Tragelaphus eurycerus isaaci, as a model system. Identification accuracy was assessed with a matching task that required same/different decisions to side-by-side pairings of individual bongos. Error rates were lowest when only the flanks of bongos were shown, suggesting that the inclusion of other visual traits confounded accuracy. Accuracy was also higher for photographs of captive animals than camera-trap images, and in observers experienced in working with mountain bongos, than those unfamiliar with the sub-species. These results suggest that the removal of non-essential morphological traits from photographs of bongos, the use of high-quality images, and relevant expertise all help increase identification accuracy. Finally, given the rise in automated identification and the use of citizen science, something our results would suggest is applicable within the context of the mountain bongo, this study provides a framework for assessing their accuracy in individual as well as species identification. PMID:26587336

  7. Factors affecting the identification of individual mountain bongo antelope

    PubMed Central

    Bindemann, Markus; Roberts, David L.

    2015-01-01

    The recognition of individuals forms the basis of many endangered species monitoring protocols. This process typically relies on manual recognition techniques. This study aimed to calculate a measure of the error rates inherent within the manual technique and also sought to identify visual traits that aid identification, using the critically endangered mountain bongo, Tragelaphus eurycerus isaaci, as a model system. Identification accuracy was assessed with a matching task that required same/different decisions to side-by-side pairings of individual bongos. Error rates were lowest when only the flanks of bongos were shown, suggesting that the inclusion of other visual traits confounded accuracy. Accuracy was also higher for photographs of captive animals than camera-trap images, and in observers experienced in working with mountain bongos, than those unfamiliar with the sub-species. These results suggest that the removal of non-essential morphological traits from photographs of bongos, the use of high-quality images, and relevant expertise all help increase identification accuracy. Finally, given the rise in automated identification and the use of citizen science, something our results would suggest is applicable within the context of the mountain bongo, this study provides a framework for assessing their accuracy in individual as well as species identification. PMID:26587336

  8. HLA-DMA polymorphisms differentially affect MHC class II peptide loading.

    PubMed

    Álvaro-Benito, Miguel; Wieczorek, Marek; Sticht, Jana; Kipar, Claudia; Freund, Christian

    2015-01-15

    During the adaptive immune response, MHCII proteins display antigenic peptides on the cell surface of APCs for CD4(+) T cell surveillance. HLA-DM, a nonclassical MHCII protein, acts as a peptide exchange catalyst for MHCII, editing the peptide repertoire. Although they map to the same gene locus, MHCII proteins exhibit a high degree of polymorphism, whereas only low variability has been observed for HLA-DM. As HLA-DM activity directly favors immunodominant peptide presentation, polymorphisms in HLA-DM (DMA or DMB chain) might well be a contributing risk factor for autoimmunity and immune disorders. Our systematic comparison of DMA*0103/DMB*0101 (DMA-G155A and DMA-R184H) with DMA*0101/DMB*0101 in terms of catalyzed peptide exchange and dissociation, as well as direct interaction with several HLA-DR/peptide complexes, reveals an attenuated catalytic activity of DMA*0103/DMB*0101. The G155A substitution dominates the catalytic behavior of DMA*0103/DMB*0101 by decreasing peptide release velocity. Preloaded peptide-MHCII complexes exhibit ∼2-fold increase in half-life in the presence of DMA*0103/DMB*0101 when compared with DMA*0101/DMB*0101. We show that this effect leads to a greater persistence of autoimmunity-related Ags in the presence of high-affinity competitor peptide. Our study therefore reveals that HLA-DM polymorphic residues have a considerable impact on HLA-DM catalytic activity.

  9. Antimicrobial peptides

    PubMed Central

    2014-01-01

    With increasing antibiotics resistance, there is an urgent need for novel infection therapeutics. Since antimicrobial peptides provide opportunities for this, identification and optimization of such peptides have attracted much interest during recent years. Here, a brief overview of antimicrobial peptides is provided, with focus placed on how selected hydrophobic modifications of antimicrobial peptides can be employed to combat also more demanding pathogens, including multi-resistant strains, without conferring unacceptable toxicity. PMID:24758244

  10. Aging Affects Identification of Vocal Emotions in Semantically Neutral Sentences

    ERIC Educational Resources Information Center

    Dupuis, Kate; Pichora-Fuller, M. Kathleen

    2015-01-01

    Purpose: The authors determined the accuracy of younger and older adults in identifying vocal emotions using the Toronto Emotional Speech Set (TESS; Dupuis & Pichora-Fuller, 2010a) and investigated the possible contributions of auditory acuity and suprathreshold processing to emotion identification accuracy. Method: In 2 experiments, younger…

  11. On the Environmental Factors Affecting the Structural and Cytotoxic Properties of IAPP Peptides

    PubMed Central

    Tomasello, Marianna Flora; Sinopoli, Alessandro; Pappalardo, Giuseppe

    2015-01-01

    Pancreatic islets in type 2 diabetes mellitus (T2DM) patients are characterized by reduced β-cells mass and diffuse extracellular amyloidosis. Amyloid deposition involves the islet amyloid polypeptide (IAPP), a neuropancreatic hormone cosecreted with insulin by β-cells. IAPP is physiologically involved in glucose homeostasis, but it may turn toxic to β-cells owing to its tendency to misfold giving rise to oligomers and fibrils. The process by which the unfolded IAPP starts to self-assemble and the overall factors promoting this conversion are poorly understood. Other open questions are related to the nature of the IAPP toxic species and how exactly β-cells die. Over the last decades, there has been growing consensus about the notion that early molecular assemblies, notably small hIAPP oligomers, are the culprit of β-cells decline. Numerous environmental factors might affect the conformational, aggregation, and cytotoxic properties of IAPP. Herein we review recent progress in the field, focusing on the influences that membranes, pH, and metal ions may have on the conformational conversion and cytotoxicity of full-length IAPP as well as peptide fragments thereof. Current theories proposed for the mechanisms of toxicity will be also summarized together with an outline of the underlying molecular links between IAPP and amyloid beta (Aβ) misfolding. PMID:26582441

  12. Factors affecting circulating levels of peptide YY in humans: a comprehensive review.

    PubMed

    Cooper, Jamie A

    2014-06-01

    As obesity continues to be a global epidemic, research into the mechanisms of hunger and satiety and how those signals act to regulate energy homeostasis persists. Peptide YY (PYY) is an acute satiety signal released upon nutrient ingestion and has been shown to decrease food intake when administered exogenously. More recently, investigators have studied how different factors influence PYY release and circulating levels in humans. Some of these factors include exercise, macronutrient composition of the diet, body-weight status, adiposity levels, sex, race and ageing. The present article provides a succinct and comprehensive review of the recent literature published on the different factors that influence PYY release and circulating levels in humans. Where human data are insufficient, evidence in animal or cell models is summarised. Additionally, the present review explores the recent findings on PYY responses to different dietary fatty acids and how this new line of research will make an impact on future studies on PYY. Human demographics, such as sex and age, do not appear to influence PYY levels. Conversely, adiposity or BMI, race and acute exercise all influence circulating PYY levels. Both dietary fat and protein strongly stimulate PYY release. Furthermore, MUFA appear to result in a smaller PYY response compared with SFA and PUFA. PYY levels appear to be affected by acute exercise, macronutrient composition, adiposity, race and the composition of fatty acids from dietary fat.

  13. Identification of Quorum-Sensing Inhibitors Disrupting Signaling between Rgg and Short Hydrophobic Peptides in Streptococci

    PubMed Central

    Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Lee, Hyun; Chlipala, George E.; Ratia, Kiira

    2015-01-01

    ABSTRACT Bacteria coordinate a variety of social behaviors, important for both environmental and pathogenic bacteria, through a process of intercellular chemical signaling known as quorum sensing (QS). As microbial resistance to antibiotics grows more common, a critical need has emerged to develop novel anti-infective therapies, such as an ability to attenuate bacterial pathogens by means of QS interference. Rgg quorum-sensing pathways, widespread in the phylum Firmicutes, employ cytoplasmic pheromone receptors (Rgg transcription factors) that directly bind and elicit gene expression responses to imported peptide signals. In the human-restricted pathogen Streptococcus pyogenes, the Rgg2/Rgg3 regulatory circuit controls biofilm development in response to the short hydrophobic peptides SHP2 and SHP3. Using Rgg-SHP as a model receptor-ligand target, we sought to identify chemical compounds that could specifically inhibit Rgg quorum-sensing circuits. Individual compounds from a diverse library of known drugs and drug-like molecules were screened for their ability to disrupt complexes of Rgg and FITC (fluorescein isothiocyanate)-conjugated SHP using a fluorescence polarization (FP) assay. The best hits were found to bind Rgg3 in vitro with submicromolar affinities, to specifically abolish transcription of Rgg2/3-controlled genes, and to prevent biofilm development in S. pyogenes without affecting bacterial growth. Furthermore, the top hit, cyclosporine A, as well as its nonimmunosuppressive analog, valspodar, inhibited Rgg-SHP pathways in multiple species of Streptococcus. The Rgg-FITC-peptide-based screen provides a platform to identify inhibitors specific for each Rgg type. Discovery of Rgg inhibitors constitutes a step toward the goal of manipulating bacterial behavior for purposes of improving health. PMID:25968646

  14. Optimization of hydrolysis conditions, isolation, and identification of neuroprotective peptides derived from seahorse Hippocampus trimaculatus.

    PubMed

    Pangestuti, Ratih; Ryu, Bomi; Himaya, Swa; Kim, Se-Kwon

    2013-08-01

    Hippocampus trimaculatus is one of the most heavily traded seahorse species for traditional medicine purposes in many countries. In the present study, we showed neuroprotective effects of peptide derived from H. trimaculatus against amyloid-β42 (Aβ42) toxicity which are central to the pathogenesis of Alzheimer's diseases (AD). Firstly, H. trimaculatus was separately hydrolyzed by four different enzymes and tested for their protective effect on Aβ42-induced neurotoxicity in differentiated PC12 cells. Pronase E hydrolysate exerted highest protection with cell viability value of 88.33 ± 3.33 %. Furthermore, we used response surface methodology to optimize pronase E hydrolysis conditions and found that temperature at 36.69 °C with the hydrolysis time 20.01 h, enzyme to substrate (E/S) ratio of 2.02 % and pH 7.34 were the most optimum conditions. Following several purification steps, H. trimaculatus-derived neuroprotective peptides (HTP-1) sequence was identified as Gly-Thr-Glu-Asp-Glu-Leu-Asp-Lys (906.4 Da). HTP-1 protected PC12 cells from Aβ42-induced neuronal death with the cell viability value of 85.52 ± 2.22 % and up-regulated pro-survival gene (Bcl-2) expressions. These results suggest that HTP-1 has the potential to be used in treatment of neurodegenerative diseases, particularly AD. Identification, characterization, and synthesis of bioactive components derived from H. trimaculatus have the potential to replace or at least complement the use of seahorse as traditional medicine, which further may become an approach to minimize seahorse exploitation in traditional medicine.

  15. Origin and evolution of peptide-modifying dioxygenases and identification of the wybutosine hydroxylase/hydroperoxidase

    PubMed Central

    Iyer, Lakshminarayan M.; Abhiman, Saraswathi; de Souza, Robson F.; Aravind, L.

    2010-01-01

    Unlike classical 2-oxoglutarate and iron-dependent dioxygenases, which include several nucleic acid modifiers, the structurally similar jumonji-related dioxygenase superfamily was only known to catalyze peptide modifications. Using comparative genomics methods, we predict that a family of jumonji-related enzymes catalyzes wybutosine hydroxylation/peroxidation at position 37 of eukaryotic tRNAPhe. Identification of this enzyme raised questions regarding the emergence of protein- and nucleic acid-modifying activities among jumonji-related domains. We addressed these with a natural classification of DSBH domains and reconstructed the precursor of the dioxygenases as a sugar-binding domain. This precursor gave rise to sugar epimerases and metal-binding sugar isomerases. The sugar isomerase active site was exapted for catalysis of oxygenation, with a radiation of these enzymes in bacteria, probably due to impetus from the primary oxygenation event in Earth’s history. 2-Oxoglutarate-dependent versions appear to have further expanded with rise of the tricarboxylic acid cycle. We identify previously under-appreciated aspects of their active site and multiple independent innovations of 2-oxoacid-binding basic residues among these superfamilies. We show that double-stranded β-helix dioxygenases diversified extensively in biosynthesis and modification of halogenated siderophores, antibiotics, peptide secondary metabolites and glycine-rich collagen-like proteins in bacteria. Jumonji-related domains diversified into three distinct lineages in bacterial secondary metabolism systems and these were precursors of the three major clades of eukaryotic enzymes. The specificity of wybutosine hydroxylase/peroxidase probably relates to the structural similarity of the modified moiety to the ancestral amino acid substrate of this superfamily. PMID:20423905

  16. Identification of Action Units Related to Affective States in a Tutoring System for Mathematics

    ERIC Educational Resources Information Center

    Padrón-Rivera, Gustavo; Rebolledo-Mendez, Genaro; Parra, Pilar Pozos; Huerta-Pacheco, N. Sofia

    2016-01-01

    Affect is an important element of the learning process both in the classroom and with educational technology. This paper presents analyses in relation to the identification of Action Units (AUs) related to affective states and their impact on learning with a tutoring system. To assess affect, a tool was devised to identify AUs on pictures of human…

  17. The chain length of biologically produced (R)-3-hydroxyalkanoic acid affects biological activity and structure of anti-cancer peptides.

    PubMed

    Szwej, Emilia; Devocelle, Marc; Kenny, Shane; Guzik, Maciej; O'Connor, Stephen; Nikodinovic-Runic, Jasmina; Radivojevic, Jelena; Maslak, Veselin; Byrne, Annete T; Gallagher, William M; Zulian, Qun Ren; Zinn, Manfred; O'Connor, Kevin E

    2015-06-20

    Conjugation of DP18L peptide with (R)-3-hydroxydecanoic acid, derived from the biopolymer polyhydroxyalkanoate, enhances its anti-cancer activity (O'Connor et al., 2013. Biomaterials 34, 2710-2718). However, it is unknown if other (R)-3-hydroxyalkanoic acids (R3HAs) can enhance peptide activity, if chain length affects enhancement, and what effect R3HAs have on peptide structure. Here we show that the degree of enhancement of peptide (DP18L) anti-cancer activity by R3HAs is carbon chain length dependent. In all but one example the R3HA conjugated peptides were more active against cancer cells than the unconjugated peptides. However, R3HAs with 9 and 10 carbons were most effective at improving DP18L activity. DP18L peptide variant DP17L, missing a hydrophobic amino acid (leucine residue 4) exhibited lower efficacy against MiaPaCa cells. Circular dichroism analysis showed DP17L had a lower alpha helix content and the conjugation of any R3HA ((R)-3-hydroxyhexanoic acid to (R)-3-hydroxydodecanoic acid) to DP17L returned the helix content back to levels of DP18L. However (R)-3-hydroxyhexanoic did not enhance the anti-cancer activity of DP17L and at least 7 carbons were needed in the R3HA to enhance activity of D17L. DP17L needs a longer chain R3HA to achieve the same activity as DP18L conjugated to an R3HA. As a first step to assess the synthetic potential of polyhydroxyalkanoate derived R3HAs, (R)-3-hydroxydecanoic acid was synthetically converted to (±)3-chlorodecanoic acid, which when conjugated to DP18L improved its antiproliferative activity against MiaPaCa cells.

  18. Identification and mapping of linear antibody epitopes in human serum albumin using high-density Peptide arrays.

    PubMed

    Hansen, Lajla Bruntse; Buus, Soren; Schafer-Nielsen, Claus

    2013-01-01

    We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm(2). Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level.

  19. Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae.

    PubMed

    Iwakoshi-Ukena, Eiko; Ukena, Kazuyoshi; Okimoto, Aiko; Soga, Miyuki; Okada, Genya; Sano, Naomi; Fujii, Tamotsu; Sugawara, Yoshiaki; Sumida, Masayuki

    2011-04-01

    The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus. PMID:21193000

  20. 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

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

    PubMed

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

    2013-01-01

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

  2. A Rapid and Simple LC-MS Method Using Collagen Marker Peptides for Identification of the Animal Source of Leather.

    PubMed

    Kumazawa, Yuki; Taga, Yuki; Iwai, Kenji; Koyama, Yoh-Ichi

    2016-08-01

    Identification of the animal source of leather is difficult using traditional methods, including microscopic observation and PCR. In the present study, a LC-MS method was developed for detecting interspecies differences in the amino acid sequence of type I collagen, which is a major component of leather, among six animals (cattle, horse, pig, sheep, goat, and deer). After a dechroming procedure and trypsin digestion, six tryptic peptides of type I collagen were monitored by LC-MS in multiple reaction monitoring mode for the animal source identification using the patterns of the presence or absence of the marker peptides. We analyzed commercial leathers from various production areas using this method, and found some leathers in which the commercial label disagreed with the identified animal source. Our method enabled rapid and simple leather certification and could be applied to other animals whether or not their collagen sequences are available in public databases.

  3. A Rapid and Simple LC-MS Method Using Collagen Marker Peptides for Identification of the Animal Source of Leather.

    PubMed

    Kumazawa, Yuki; Taga, Yuki; Iwai, Kenji; Koyama, Yoh-Ichi

    2016-08-01

    Identification of the animal source of leather is difficult using traditional methods, including microscopic observation and PCR. In the present study, a LC-MS method was developed for detecting interspecies differences in the amino acid sequence of type I collagen, which is a major component of leather, among six animals (cattle, horse, pig, sheep, goat, and deer). After a dechroming procedure and trypsin digestion, six tryptic peptides of type I collagen were monitored by LC-MS in multiple reaction monitoring mode for the animal source identification using the patterns of the presence or absence of the marker peptides. We analyzed commercial leathers from various production areas using this method, and found some leathers in which the commercial label disagreed with the identified animal source. Our method enabled rapid and simple leather certification and could be applied to other animals whether or not their collagen sequences are available in public databases. PMID:27397145

  4. The Fusion Protein Signal-Peptide-Coding Region of Canine Distemper Virus: A Useful Tool for Phylogenetic Reconstruction and Lineage Identification

    PubMed Central

    Sarute, Nicolás; Calderón, Marina Gallo; Pérez, Ruben; La Torre, José; Hernández, Martín; Francia, Lourdes; Panzera, Yanina

    2013-01-01

    Canine distemper virus (CDV; Paramyxoviridae, Morbillivirus) is the etiologic agent of a multisystemic infectious disease affecting all terrestrial carnivore families with high incidence and mortality in domestic dogs. Sequence analysis of the hemagglutinin (H) gene has been widely employed to characterize field strains, permitting the identification of nine CDV lineages worldwide. Recently, it has been established that the sequences of the fusion protein signal-peptide (Fsp) coding region are extremely variable, suggesting that analysis of its sequence might be useful for strain characterization studies. However, the divergence of Fsp sequences among worldwide strains and its phylogenetic resolution has not yet been evaluated. We constructed datasets containing the Fsp-coding region and H gene sequences of the same strains belonging to eight CDV lineages. Both datasets were used to evaluate their phylogenetic resolution. The phylogenetic analysis revealed that both datasets clustered the same strains into eight different branches, corresponding to CDV lineages. The inter-lineage amino acid divergence was fourfold greater for the Fsp peptide than for the H protein. The likelihood mapping revealed that both datasets display strong phylogenetic signals in the region of well-resolved topologies. These features indicate that Fsp-coding region sequence analysis is suitable for evolutionary studies as it allows for straightforward identification of CDV lineages. PMID:23675493

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2015-02-01

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

  7. Genome-wide identification and in silico analysis of poplar peptide deformylases.

    PubMed

    Liu, Chang-Cai; Liu, Bao-Guang; Yang, Zhi-Wei; Li, Chun-Ming; Wang, Bai-Chen; Yang, Chuan-Ping

    2012-01-01

    Peptide deformylases (PDF) behave as monomeric metal cation hydrolases for the removal of the N-formyl group (Fo). This is an essential step in the N-terminal Met excision (NME) that occurs in these proteins from eukaryotic mitochondria or chloroplasts. Although PDFs have been identified and their structure and function have been characterized in several herbaceous species, it remains as yet unexplored in poplar. Here, we report on the first identification of two genes (PtrPDF1A and PtrPDF1B) respectively encoding two putative PDF polypeptides in Populus trichocarpa by genome-wide investigation. One of them (XP_002300047.1) encoded by PtrPDF1B (XM_002300011.1) was truncated, and then revised into a complete sequence based on its ESTs support with high confidence. We document that the two PDF1s of Populus are evolutionarily divergent, likely as a result of independent duplicated events. Furthermore, in silico simulations demonstrated that PtrPDF1A and PtrPDF1B should act as similar PDF catalytic activities to their corresponding PDF orthologs in Arabidopsis. This result would be value of for further assessment of their biological activities in poplar, and further experiments are now required to confirm them. PMID:22606033

  8. High-Throughput Identification of Inhibitors of Human Mitochondrial Peptide Deformylase

    PubMed Central

    ANTCZAK, CHRISTOPHE; SHUM, DAVID; ESCOBAR, SINDY; BASSIT, BHRAMDEO; KIM, EARL; SESHAN, VENKATRAMAN E.; WU, NIAN; YANG, GUANGLI; OUERFELLI, OUATHEK; LI, YUE-MING; SCHEINBERG, DAVID A.; DJABALLAH, HAKIM

    2008-01-01

    The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid–based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)–based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent. PMID:17435169

  9. Stable isotope labeling tandem mass spectrometry (SILT): integration with peptide identification and extension to data-dependent scans.

    PubMed

    Elbert, Donald L; Mawuenyega, Kwasi G; Scott, Evan A; Wildsmith, Kristin R; Bateman, Randall J

    2008-10-01

    Quantitation of relative or absolute amounts of proteins by mass spectrometry can be prone to large errors. The use of MS/MS ion intensities and stable isotope labeling, which we term stable isotope labeling tandem mass spectrometry (SILT), decreases the effects of contamination from unrelated compounds. We present a software package (SILTmass) that automates protein identification and quantification by the SILT method. SILTmass has the ability to analyze the kinetics of protein turnover, in addition to relative and absolute protein quantitation. Instead of extracting chromatograms to find elution peaks, SILTmass uses only scans in which a peptide is identified and that meet an ion intensity threshold. Using only scans with identified peptides, the accuracy and precision of SILT is shown to be superior to precursor ion intensities, particularly at high or low dilutions of the isotope labeled compounds or with low amounts of protein. Using example scans, we demonstrate likely reasons for the improvements in quantitation by SILT. The appropriate use of variable modifications in peptide identification is described for measurement of protein turnover kinetics. The combination of identification with SILT facilitates quantitation without peak detection and helps to ensure the appropriate use of variable modifications for kinetics experiments.

  10. Identification of multiple antimicrobial peptides from the skin of fine-spined frog, Hylarana spinulosa (Ranidae).

    PubMed

    Yang, Xiaolong; Hu, Yuhong; Xu, Shiqi; Hu, Yonghong; Meng, Hao; Guo, Chao; Liu, Yuliang; Liu, Jingze; Yu, Zhijun; Wang, Hui

    2013-12-01

    In this study, peptidomics and genomics analyses were used to study antimicrobial peptides from the skin of Hylarana spinulosa. Twenty-nine different antimicrobial peptide precursors were characterized from the skin of H. spinulosa, which produce 23 mature antimicrobial peptides belonging to 12 different families. To confirm the actual presence and characteristics of these antimicrobial peptides in the skin tissue extractions from H. spinulosa, we used two distinct methods, one was peptide purification method that combined gel filtration chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), and the other was peptidomics approach based on liquid chromatography in conjunction with tandem mass spectrometry (LC-MS/MS). In the peptidomics approach, incomplete tryptic digestion and gas-phase fractionation (GPF) analysis were used to increase peptidome coverage and reproducibility of peptide ion selection. Multiple species of microorganisms were chosen to test and analyze the antimicrobial activities and spectrum of these antimicrobial peptides.

  11. Identification of five different Patr class I molecules that bind HLA supertype peptides and definition of their peptide binding motifs.

    PubMed

    McKinney, D M; Erickson, A L; Walker, C M; Thimme, R; Chisari, F V; Sidney, J; Sette, A

    2000-10-15

    We have sequenced the Pan troglodytes class I (Patr) molecules from three common chimpanzees and expressed them as single molecules in a class I-deficient cell line. These lines were utilized to obtain purified class I molecules to define the peptide binding motifs associated with five different Patr molecules. Based on these experiments, as well as analysis of the predicted structure of the B and F polymorphic MHC pockets, we classified five Patr molecules (Patr-A*0101, Patr-B*0901, Patr-B*0701, Patr-A*0602, and Patr-B*1301) within previously defined supertype specificities associated with HLA class I molecules (HLA-A3, -B7, -A1, and -A24 supertypes). The overlap in the binding repertoire between specific HLA and Patr class I molecules was in the range of 33 to 92%, depending on the particular Patr molecule as assessed by the binding of HIV-, hepatitis B virus-, and hepatitis C virus-derived epitopes. Finally, live cell binding assays of nine chimpanzee-derived B cell lines demonstrated that HLA supertype peptides bound to Patr class I molecules with frequencies in the 20-50% range. PMID:11035079

  12. Identification of a peptide specifically targeting ovarian cancer by the screening of a phage display peptide library

    PubMed Central

    WANG, LEDAN; HU, YUE; LI, WENJU; WANG, FAN; LU, XIAOSHENG; HAN, XUEYING; LV, JIEQIANG; CHEN, JIE

    2016-01-01

    Ovarian cancer is the most common cause of cancer-associated mortality in terms of gynecological malignancies, and is difficult to diagnose due to the absence of reliable biomarkers. To identify ovarian cancer-specific biomarkers, the present study used a Ph.D.-7™ Phage Display Peptide Library to screen for ligands that selectively target HO-8910 ovarian cancer cells. Following 5 rounds of biopanning, the phage clone P2 was selected by enzyme-linked immunosorbent assay and DNA sequencing, and its characteristics were additionally validated by immunofluorescence and immunohistochemical assays. The results revealed the positive phage were enriched 92-fold following 5 rounds of biopanning, and the DNA sequence AAC CCG ATG ATT CGC CGC CAG (amino acid sequence, NPMIRRQ) was repeated most frequently (phage clones, P2, P3, P15, P30 and P54). Immunofluorescence and immunohistochemical assays revealed that the phage clone P2 was able to bind to ovarian cancer cells and tissues, and not those of cervical cancer. In conclusion, the peptide NPMIRRQ may be a potential agent for the diagnosis of ovarian cancer. PMID:27313733

  13. Identification of a glycogenolysis-inhibiting peptide from the corpora cardiaca of locusts.

    PubMed

    Clynen, Elke; Huybrechts, Jurgen; Baggerman, Geert; Van Doorn, Jan; Van Der Horst, Dick; De Loof, Arnold; Schoofs, Liliane

    2003-08-01

    A mass spectrometric study of the peptidome of the neurohemal part of the corpora cardiaca of Locusta migratoria and Schistocerca gregaria shows that it contains several unknown peptides. We were able to identify the sequence of one of these peptides as pQSDLFLLSPK. This sequence is identical to the part of the Locusta insulin-related peptide (IRP) precursor that is situated between the signal peptide and the B-chain. We designated this peptide as IRP copeptide. This IRP copeptide is also present in the pars intercerebralis, which is likely to be the site of synthesis. It is identical in both L. migratoria and S. gregaria. It shows no effect on the hemolymph lipid concentration in vivo or muscle contraction in vitro. The IRP copeptide is able to cause a decreased phosphorylase activity in locust fat body in vitro, opposite to the effect of the adipokinetic hormones and therefore possibly represents a glycogenolysis-inhibiting peptide. PMID:12865323

  14. Identification of Synthetic and Natural Host Defense Peptides with Leishmanicidal Activity

    PubMed Central

    Marr, A. K.; Cen, S.; Hancock, R. E. W.

    2016-01-01

    Leishmania parasites are a major public health problem worldwide. Effective treatment of leishmaniasis is hampered by the high incidence of adverse effects to traditional drug therapy and the emergence of resistance to current therapeutics. A vaccine is currently not available. Host defense peptides have been investigated as novel therapeutic agents against a wide range of pathogens. Here we demonstrate that the antimicrobial peptide LL-37 and the three synthetic peptides E6, L-1018, and RI-1018 exhibit leishmanicidal activity against promastigotes and intramacrophage amastigotes of Leishmania donovani and Leishmania major. We also report that the Leishmania protease/virulence factor GP63 confers protection to Leishmania from the cytolytic properties of all l-form peptides (E6, L-1018, and LL-37) but not the d-form peptide RI-1018. The results suggest that RI-1018, E6, and LL-37 are promising peptides to develop further into components for antileishmanial therapy. PMID:26883699

  15. Purification and identification of endogenous antioxidant and ACE-inhibitory peptides from donkey milk by multidimensional liquid chromatography and nanoHPLC-high resolution mass spectrometry.

    PubMed

    Zenezini Chiozzi, Riccardo; Capriotti, Anna Laura; Cavaliere, Chiara; La Barbera, Giorgia; Piovesana, Susy; Samperi, Roberto; Laganà, Aldo

    2016-08-01

    Donkey milk is a valuable product for the food industry due to its nutraceutical, nutritional, and functional properties. In this work, the endogenous peptides from donkey milk were investigated for their antioxidant and ACE-inhibitory activities, combining a two-dimensional peptide fractionation strategy with high-resolution mass spectrometry, bioinformatics analysis, and in vitro assays. After extraction, the endogenous peptides were fractionated twice, first by polymeric reversed phase and then by hydrophilic interaction chromatography. Fractions were screened for the investigated bioactivities and only the most active ones were finally analyzed by nanoRP-HPLC-MS/MS; this approach allowed to reduce the total number of possible bioactive sequences. Results were further mined by in silico analysis using PeptideRanker, BioPep, and PepBank, which provided a bioactivity score to the identified peptides and matched sequences to known bioactive peptides, in order to select candidates for chemical synthesis. Thus, five peptides were prepared and then compared to the natural occurring ones, checking their retention times and fragmentation patterns in donkey milk alone and in spiked donkey milk samples. Pure peptide standards were finally in vitro tested for the specific bioactivity. In this way, two novel endogenous antioxidant peptides, namely EWFTFLKEAGQGAKDMWR and GQGAKDMWR, and two ACE-inhibitory peptides, namely REWFTFLK and MPFLKSPIVPF, were successfully validated from donkey milk. Graphical Abstract Analytical workflow for purification and identification of bioactive peptides from donkey milk sample.

  16. Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

    PubMed Central

    Kim, Hyun-Joong

    2014-01-01

    Candida albicans is an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiating C. albicans from other Candida species are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel of C. albicans and various nontarget Candida spp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power for C. albicans than P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification of C. albicans in clinical and related applications, especially when combined with FCM. PMID:25428160

  17. LC-MS/MS Identification of a Bromelain Peptide Biomarker from Ananas comosus Merr.

    PubMed

    Secor, Eric R; Szczepanek, Steven M; Singh, Anurag; Guernsey, Linda; Natarajan, Prabitha; Rezaul, Karim; Han, David K; Thrall, Roger S; Silbart, Lawrence K

    2012-01-01

    Bromelain (Br) is a cysteine peptidase (GenBank AEH26024.1) from pineapple, with over 40 years of clinical use. The constituents mediating its anti-inflammatory activity are not thoroughly characterized and no peptide biomarker exists. Our objective is to characterize Br raw material and identify peptides in the plasma of Br treated mice. After SDS-PAGE in-gel digestion, Br (VN#3507; Middletown, CT, USA) peptides were analyzed via LC/MS/MS using 95% protein probability, 95% peptide probability, and a minimum peptide number = 5. Br spiked mouse plasma (1 ug/ul) and plasma from i.p. treated mice (12 mg/kg) were assessed using SRM. In Br raw material, we identified seven proteins: four proteases, one jacalin-like lectin, and two protease inhibitors. In Br spiked mouse plasma, six proteins (ananain, bromelain inhibitor, cysteine proteinase AN11, FB1035 precursor, FBSB precursor, and jacalin-like lectin) were identified. Using LC/MS/MS, we identified the unique peptide, DYGAVNEVK, derived from FB1035, in the plasma of i.p. Br treated mice. The spectral count of this peptide peaked at 6 hrs and was undetectable by 24 hrs. In this study, a novel Br peptide was identified in the plasma of treated mice for the first time. This Br peptide could serve as a biomarker to standardize the therapeutic dose and maximize clinical utility.

  18. Can the Direct Peptide Reactivity Assay Be Used for the Identification of Respiratory Sensitization Potential of Chemicals?

    PubMed

    Dik, Sander; Rorije, Emiel; Schwillens, Paul; van Loveren, Henk; Ezendam, Janine

    2016-10-01

    Prospective identification of low molecular weight respiratory sensitizers is difficult due to the current lack of adequate test methods. The direct peptide reactivity assay (DPRA) seems to be a promising method to determine the sensitization potential of chemicals because it determines the intrinsic characteristic of sensitizers to bind to proteins. It is already applied in the field of skin sensitization, and adaptation to respiratory sensitization has started recently. This article further evaluates the ability of the DPRA to predict the respiratory sensitization potential of chemicals. In addition, the added value of applying High Performance Liquid Chromatography (HPLC)-MS and measurements after 20 minutes and 24 hours of incubation was evaluated. Eighteen respiratory sensitizers (10 haptens, 3 prehaptens, and 5 prohaptens) and 14 nonsensitizers were tested with 2-model peptides. Based on peptide depletion, a prediction model was proposed for the identification of (respiratory) sensitizers. Application of mass spectrometry and measurements at 2 time-points increased prediction accuracy of the assay by resolving discordant results. The prediction model correctly identified all haptens and prehaptens as sensitizers. The 5 prohaptens were not identified as sensitizers, most likely due to lack of metabolic activity in the DPRA. All but 1 nonsensitizer was correctly predicted. The model, therefore, shows an accuracy of 78% for the tested dataset. Unfortunately, this assay cannot be used to distinguish respiratory from skin sensitizers. To make this distinction, the DPRA needs to be combined with other test methods that are able to identify respiratory sensitizers.

  19. Overexpression of the Arabidopsis thaliana signalling peptide TAXIMIN1 affects lateral organ development

    PubMed Central

    Colling, Janine; Tohge, Takayuki; De Clercq, Rebecca; Brunoud, Geraldine; Vernoux, Teva; Fernie, Alisdair R.; Makunga, Nokwanda P.; Goossens, Alain; Pauwels, Laurens

    2015-01-01

    Lateral organ boundary formation is highly regulated by transcription factors and hormones such as auxins and brassinosteroids. However, in contrast to many other developmental processes in plants, no role for signalling peptides in the regulation of this process has been reported yet. The first characterization of the secreted cysteine-rich TAXIMIN (TAX) signalling peptides in Arabidopsis is presented here. TAX1 overexpression resulted in minor alterations in the primary shoot and root metabolome, abnormal fruit morphology, and fusion of the base of cauline leaves to stems forming a decurrent leaf attachment. The phenotypes at the paraclade junction match TAX1 promoter activity in this region and are similar to loss of LATERAL ORGAN FUSION (LOF) transcription factor function. Nevertheless, TAX1 expression was unchanged in lof1lof2 paraclade junctions and, conversely, LOF gene expression was unchanged in TAX1 overexpressing plants, suggesting TAX1 may act independently. This study identifies TAX1 as the first plant signalling peptide influencing lateral organ separation and implicates the existence of a peptide signal cascade regulating this process in Arabidopsis. PMID:26071531

  20. Overexpression of the Arabidopsis thaliana signalling peptide TAXIMIN1 affects lateral organ development.

    PubMed

    Colling, Janine; Tohge, Takayuki; De Clercq, Rebecca; Brunoud, Geraldine; Vernoux, Teva; Fernie, Alisdair R; Makunga, Nokwanda P; Goossens, Alain; Pauwels, Laurens

    2015-08-01

    Lateral organ boundary formation is highly regulated by transcription factors and hormones such as auxins and brassinosteroids. However, in contrast to many other developmental processes in plants, no role for signalling peptides in the regulation of this process has been reported yet. The first characterization of the secreted cysteine-rich TAXIMIN (TAX) signalling peptides in Arabidopsis is presented here. TAX1 overexpression resulted in minor alterations in the primary shoot and root metabolome, abnormal fruit morphology, and fusion of the base of cauline leaves to stems forming a decurrent leaf attachment. The phenotypes at the paraclade junction match TAX1 promoter activity in this region and are similar to loss of LATERAL ORGAN FUSION (LOF) transcription factor function. Nevertheless, TAX1 expression was unchanged in lof1lof2 paraclade junctions and, conversely, LOF gene expression was unchanged in TAX1 overexpressing plants, suggesting TAX1 may act independently. This study identifies TAX1 as the first plant signalling peptide influencing lateral organ separation and implicates the existence of a peptide signal cascade regulating this process in Arabidopsis. PMID:26071531

  1. Identification of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Enzymatic Hydrolysates of Razor Clam Sinonovacula constricta.

    PubMed

    Li, Yun; Sadiq, Faizan A; Fu, Li; Zhu, Hui; Zhong, Minghua; Sohail, Muhammad

    2016-01-01

    Angiotensin I-converting enzyme (ACE) inhibitory activity of razor clam hydrolysates produced using five proteases, namely, pepsin, trypsin, alcalase, flavourzyme and proteases from Actinomucor elegans T3 was investigated. Flavourzyme hydrolysate showed the highest level of degree of hydrolysis (DH) (45.87%) followed by A. elegans T3 proteases hydrolysate (37.84%) and alcalase (30.55%). The A. elegans T3 proteases was observed to be more effective in generating small peptides with ACE-inhibitory activity. The 3 kDa membrane permeate of A. elegans T3 proteases hydrolysate showed the highest ACE-inhibitory activity with an IC50 of 0.79 mg/mL. After chromatographic separation by Sephadex G-15 gel filtration and reverse phase-high performance liquid chromatography, the potent fraction was subjected to MALDI/TOF-TOF MS/MS for identification. A novel ACE-inhibitory peptide (VQY) was identified exhibiting an IC50 of 9.8 μM. The inhibitory kinetics investigation by Lineweaver-Burk plots demonstrated that the peptide acts as a competitive ACE inhibitor. The razor clam hydrolysate obtained by A. elegans T3 proteases could serve as a source of functional peptides with ACE-inhibitory activity for physiological benefits. PMID:27271639

  2. Identification of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Enzymatic Hydrolysates of Razor Clam Sinonovacula constricta

    PubMed Central

    Li, Yun; Sadiq, Faizan A.; Fu, Li; Zhu, Hui; Zhong, Minghua; Sohail, Muhammad

    2016-01-01

    Angiotensin I-converting enzyme (ACE) inhibitory activity of razor clam hydrolysates produced using five proteases, namely, pepsin, trypsin, alcalase, flavourzyme and proteases from Actinomucor elegans T3 was investigated. Flavourzyme hydrolysate showed the highest level of degree of hydrolysis (DH) (45.87%) followed by A. elegans T3 proteases hydrolysate (37.84%) and alcalase (30.55%). The A. elegans T3 proteases was observed to be more effective in generating small peptides with ACE-inhibitory activity. The 3 kDa membrane permeate of A. elegans T3 proteases hydrolysate showed the highest ACE-inhibitory activity with an IC50 of 0.79 mg/mL. After chromatographic separation by Sephadex G-15 gel filtration and reverse phase-high performance liquid chromatography, the potent fraction was subjected to MALDI/TOF-TOF MS/MS for identification. A novel ACE-inhibitory peptide (VQY) was identified exhibiting an IC50 of 9.8 μM. The inhibitory kinetics investigation by Lineweaver-Burk plots demonstrated that the peptide acts as a competitive ACE inhibitor. The razor clam hydrolysate obtained by A. elegans T3 proteases could serve as a source of functional peptides with ACE-inhibitory activity for physiological benefits. PMID:27271639

  3. Effectiveness of CID, HCD, and ETD with FT MS/MS for Degradomic-Peptidomic Analysis: Comparison of Peptide Identification Methods

    SciTech Connect

    Shen, Yufeng; Tolić, Nikola; Xie, Fang; Zhao, Rui; Purvine, Samuel O.; Schepmoes, Athena A.; Moore, Ronald, J.; Anderson, Gordon A.; Smith, Richard D.

    2011-09-02

    We report on use of an Orbitrap Velos mass spectrometer for comparison of fragmentation methods namely CID-, HCD-, and ETD for FT MS/MS analysis of human blood plasma peptidomic peptides. The peptidomic peptides were able to be identified from CID, HCD, and ETD spectra on specific confidence levels (e.g., 1% false discovery rate) with use of conventional SEQUEST database search software, and the number of identified peptides was increased by ~50% using accurate fragments (e.g., with mass tolerance of 0.05Da) in comparison with traditional moderate accuracy fragments (e.g., with 1 Da mass tolerance) for database search. However, the peptide datasets identified with such decoy search strategy were found to be varied by ~25% in the dataset size and ~20% in the dataset content with type of decoy database and precursor mass tolerances used for database search. CID was evaluated as the largest contributor to the identified peptide datasets, and HCD, and ETD provided ~20% and ~22% respectively additional peptides with accurate fragments for peptide identification, in contrast to ~25% and ~13% respectively with use of moderate accuracy fragments. When long (typically ≥7 amino acids) sequences were used for identification of peptides from the previously published UStags and de novo sequencing methods, HCD was evaluated as the largest contributor, and CID and ETD provided ~26% and ~8% respectively additional peptides from the UStags method and ~26% and ~6% respectively additional peptides from the de novo sequencing method. The peptide datasets identified with the UStags method were little influenced by the decoy database and mass tolerance and 98-99% peptide overlaps could be achieved between these datasets. CID, HCD, and ETD contributed their identifications of various charge state peptides in the m/z range highly overlapped and complementary implementation of CID, HCD, and ETD should be applied to maximize the number of peptides identified. Finally, the investigation

  4. Transmissible gastroenteritis virus; identification of M protein-binding peptide ligands with antiviral and diagnostic potential

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and ...

  5. Identification of patterns in diffraction intensities affected by radiation exposure.

    PubMed

    Borek, Dominika; Dauter, Zbigniew; Otwinowski, Zbyszek

    2013-01-01

    In an X-ray diffraction experiment, the structure of molecules and the crystal lattice changes owing to chemical reactions and physical processes induced by the absorption of X-ray photons. These structural changes alter structure factors, affecting the scaling and merging of data collected at different absorbed doses. Many crystallographic procedures rely on the analysis of consistency between symmetry-equivalent reflections, so failure to account for the drift of their intensities hinders the structure solution and the interpretation of structural results. The building of a conceptual model of radiation-induced changes in macromolecular crystals is the first step in the process of correcting for radiation-induced inconsistencies in diffraction data. Here the complexity of radiation-induced changes in real and reciprocal space is analysed using matrix singular value decomposition applied to multiple complete datasets obtained from single crystals. The model consists of a resolution-dependent decay correction and a uniform-per-unique-reflection term modelling specific radiation-induced changes. This model is typically sufficient to explain radiation-induced effects observed in diffraction intensities. This analysis will guide the parameterization of the model, enabling its use in subsequent crystallographic calculations.

  6. Identification of factors affecting birth rate in Czech Republic

    NASA Astrophysics Data System (ADS)

    Zámková, Martina; Blašková, Veronika

    2013-10-01

    This article is concerned with identifying economic factors primarily that affect birth rates in Czech Republic. To find the relationship between the magnitudes, we used the multivariate regression analysis and for modeling, we used a time series of annual values (1994-2011) both economic indicators and indicators related to demographics. Due to potential problems with apparent dependence we first cleansed all series obtained from the Czech Statistical Office using first differences. It is clear from the final model that meets all assumptions that there is a positive correlation between birth rates and the financial situation of households. We described the financial situation of households by GDP per capita, gross wages and consumer price index. As expected a positive correlation was proved for GDP per capita and gross wages and negative dependence was proved for the consumer price index. In addition to these economic variables in the model there were used also demographic characteristics of the workforce and the number of employed people. It can be stated that if the Czech Republic wants to support an increase in the birth rate, it is necessary to consider the financial support for households with small children.

  7. Comprehensive Identification of Glycated Peptides and Their Glycation Motifs in Plasma and Erythrocytes of Control and Diabetic Subjects

    SciTech Connect

    Zhang, Qibin; Monroe, Matthew E.; Schepmoes, Athena A.; Clauss, Therese RW; Gritsenko, Marina A.; Meng, Da; Petyuk, Vladislav A.; Smith, Richard D.; Metz, Thomas O.

    2011-07-01

    Non-enzymatic glycation of proteins is implicated in diabetes mellitus and its related complications. In this report, we extend our previous development and refinement of proteomics-based methods for the analysis of non-enzymatically glycated proteins to comprehensively identify glycated proteins in normal and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semi-quantitative comparisons revealed a number of proteins with glycation levels significantly increased in diabetes relative to control samples and that erythrocyte proteins are more extensively glycated than plasma proteins. A glycation motif analysis revealed amino acids that are favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for the potential identification of novel markers for diabetes, glycemia, or diabetic complications.

  8. Purification and identification of novel antioxidant peptides from egg white protein and their antioxidant activities.

    PubMed

    Liu, Jingbo; Jin, Yan; Lin, Songyi; Jones, Gregory S; Chen, Feng

    2015-05-15

    The aim of this study was to isolate antioxidant peptides from egg white protein hydrolysates (EWPH) and identify novel antioxidant peptides by LC tandem mass spectrometric and mid-infrared spectroscopy (MIR). The amino acid composition of peptides was also analyzed by amino acid analyzer on the basis of ninhydrin reaction. Three novel peptides with molecular weights of 628.64 Da, 630.71 Da, and 684.1 Da were identified as Asp-His-Thr-Lys-Glu (DHTKE), Phe-Phe-Glu-Phe-His (FFGFN) and Met-Pro-Asp-Ala-His-Leu (MPDAHL), respectively. DHTKE exhibited the best oxygen radical absorbance capacity (P<0.05). The concentration of FFGFN and MPDAHL to scavenge 50% of DPPH radicals was 80 mM and 60mM, respectively. Thus, the three peptides may have potential applications as a functional food, which could also be used as nutraceutical compounds. PMID:25577078

  9. Identification of an amyloidogenic peptide from the Bap protein of Staphylococcus epidermidis.

    PubMed

    Lembré, Pierre; Vendrely, Charlotte; Martino, Patrick Di

    2014-01-01

    Biofilm associated proteins (Bap) are involved in the biofilm formation process of several bacterial species. The sequence STVTVT is present in Bap proteins expressed by many Staphylococcus species, Acinetobacter baumanii and Salmonella enterica. The peptide STVTVTF derived from the C-repeat of the Bap protein from Staphylococcus epidermidis was selected through the AGGRESCAN, PASTA, and TANGO software prediction of protein aggregation and formation of amyloid fibers. We characterized the self-assembly properties of the peptide STVTVTF by different methods: in the presence of the peptide, we observed an increase in the fluorescence intensity of Thioflavin T; many intermolecular β-sheets and fibers were spontaneously formed in peptide preparations as observed by infrared spectroscopy and atomic force microscopy analyses. In conclusion, a 7 amino acids peptide derived from the C-repeat of the Bap protein was sufficient for the spontaneous formation of amyloid fibers. The possible involvement of this amyloidogenic sequence in protein-protein interactions is discussed.

  10. Identification of B cell and T cell epitopes using synthetic peptide combinatorial libraries.

    PubMed

    Pinilla, Clemencia; Appel, Jon R; Judkowski, Valeria; Houghten, Richard A

    2012-11-01

    This unit presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries of peptide molecules. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide library to identify T cell peptide ligands. The 200 mixtures of the decapeptide library are tested for their ability to induce T cell activation. Support protocols cover optimization of the assay conditions for each MAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide library, along with deconvolution approaches.

  11. Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries.

    PubMed

    Miller, Chad J; Turk, Benjamin E

    2016-01-01

    Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors.

  12. Charge Distribution and Imperfect Amphipathicity Affect Pore Formation by Antimicrobial Peptides

    PubMed Central

    Mihajlovic, Maja

    2012-01-01

    Antimicrobial peptides often permeabilize biological membranes via a pore mechanism. Two pore types have been proposed: toroidal, where the pore is partly lined by lipid, and barrel-stave, where a cylindrical pore is completely lined by peptides. What drives the preference of antimicrobial peptides for a certain pore type is not yet fully understood. According to neutron scattering and oriented circular dichroism, melittin and MG-H2 induce toroidal pores whereas alamethicin forms barrel-stave pores. In previous work we found that indeed melittin seems to favor toroidal pores whereas alamethicin favors cylindrical pores. Here we designed mutants of these two peptides and the magainin analogue MG-H2, aimed to probe how the distribution of charges along the helix and its imperfectly amphipathic structure influence pore formation. Molecular dynamics (MD) simulations of the peptides in a pre-formed cylindrical pore have been performed. The duration of the simulations was 136 ns to 216 ns. We found that a melittin mutant with lysine 7 neutralized favors cylindrical pores whereas a MG-H2 mutant with lysines in the N-terminal half of these peptides neutralized and an alamethicin mutant with a positive charge at the position 7 form semitoroidal pores. These results suggest that charged residues within the N-terminal half are important for the toroidal pore formation. Toroidal pores produced by MG-H2 are more disordered than the melittin pores, likely because of the charged residues located in the middle of the MG-H2 helix (K11 and K14). Imperfect amphipathicity of melitin seems to play a role in its preference for toroidal pores since the substitutions of charged residues located within the nonpolar face by hydrophobic residues suppress evolution of a toroidal pore. The mutations change the position of lysine 7 near the N-terminus, relative to the lower leaflet headgroups. The MD simulations also show that the melittin P14A mutant forms a toroidal pore, but its

  13. Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma.

    PubMed

    Chai, San Jiun; Yap, Yoke Yeow; Foo, Yoke Ching; Yap, Lee Fah; Ponniah, Sathibalan; Teo, Soo Hwang; Cheong, Sok Ching; Patel, Vyomesh; Lim, Kue Peng

    2015-01-01

    Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9-20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients.

  14. Identification of a peptide binding protein that plays a role in antigen presentation

    SciTech Connect

    Lakey, E.K.; Margoliash, E.; Pierce, S.K.

    1987-03-01

    The helper T-cell response to globular proteins appears, in general, to require intracellular processing of the antigen, such that a peptide fragment containing the T-cell antigenic determinant is released and transported to and held on the surface of an Ia-expressing, antigen-presenting cell. However, the molecular details underlying these phenomena are largely unknown. The means by which antigenic peptides are anchored on the antigen-presenting cell surface was investigated. A cell surface protein is identified that was isolated by it ability to bind to a 24-amino acid peptide fragment of pigeon cytochrome c, residues 81-104, containing the major antigenic determinant for B10.A mouse T cells. This peptide binding protein, purified from (/sup 35/S)methionine-labeled cells, appears as two discrete bands of approx. =72 and 74 kDa after NaDodSO/sub 4//PAGE. The protein can be eluted from the peptide affinity column with equivalent concentrations of either the antigenic pigeon cytochrome c peptide or the corresponding nonantigenic peptide of mouse cytochrome c. However, it does not bind to the native cytochromes c, either of pigeon or mouse, and thus the protein appears to recognize some structure available only in the free peptides. This protein plays a role in antigen presentation. Its expression is not major histocompatibility complex-restricted in that the blocking activity of the antisera can be absorbed on spleen cells from mice of different haplotypes. This peptide binding protein can be isolated from a variety of cell types, including B cells, T cells, and fibroblasts. The anchoring of processed peptides on the cell surface by such a protein may play a role in antigen presentation.

  15. Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma

    PubMed Central

    Chai, San Jiun; Yap, Yoke Yeow; Foo, Yoke Ching; Yap, Lee Fah; Ponniah, Sathibalan; Teo, Soo Hwang; Cheong, Sok Ching; Patel, Vyomesh; Lim, Kue Peng

    2015-01-01

    Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9–20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients. PMID:26536470

  16. Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma.

    PubMed

    Chai, San Jiun; Yap, Yoke Yeow; Foo, Yoke Ching; Yap, Lee Fah; Ponniah, Sathibalan; Teo, Soo Hwang; Cheong, Sok Ching; Patel, Vyomesh; Lim, Kue Peng

    2015-01-01

    Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9-20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients. PMID:26536470

  17. Identification of novel dipeptidyl peptidase-IV and angiotensin-I-converting enzyme inhibitory peptides from meat proteins using in silico analysis.

    PubMed

    Lafarga, Tomas; O'Connor, Paula; Hayes, Maria

    2014-09-01

    Angiotensin-I-converting enzyme (ACE-I, EC 3.4.15.1), renin (EC 3.4.23.15), and dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5) play key roles in the control of hypertension and the development of type-2 diabetes and other diseases associated with metabolic syndrome. The aim of this work was to utilize known in silico methodologies, peptide databases and software including ProtParam (http://web.expasy.org/protparam/), Basic Local Alignment Tool (BLAST), ExPASy PeptideCutter (http://web.expasy.org/peptide_cutter/) and BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep) to assess the release of potentially bioactive DPP-IV, renin and ACE-I inhibitory peptides from bovine and porcine meat proteins including hemoglobin, collagen and serum albumin. These proteins were chosen as they are found commonly in meat by-products such as bone, blood and low-value meat cuts. In addition, the bioactivities of identified peptides were confirmed using chemical synthesis and in vitro bioassays. The concentration of peptide required to inhibit the activity of ACE-I and DPP-IV by 50% was determined for selected, active peptides. Novel ACE-I and DPP-IV inhibitory peptides were identified in this study using both in silico analysis and a literature search to streamline enzyme selection for peptide production. These novel peptides included the ACE-I inhibitory tri-peptide Ile-Ile-Tyr and the DPP-IV inhibitory tri-peptide Pro-Pro-Leu corresponding to sequences f (182-184) and f (326-328) of both porcine and bovine serum albumin which can be released following hydrolysis with the enzymes papain and pepsin, respectively. This work demonstrates that meat proteins are a suitable resource for the generation of bioactive peptides and further demonstrates the usefulness of in silico methodologies to streamline identification and generation of bioactive peptides.

  18. The major genetic determinants of HIV-1 control affect HLA class I peptide presentation.

    PubMed

    Pereyra, Florencia; Jia, Xiaoming; McLaren, Paul J; Telenti, Amalio; de Bakker, Paul I W; Walker, Bruce D; Ripke, Stephan; Brumme, Chanson J; Pulit, Sara L; Carrington, Mary; Kadie, Carl M; Carlson, Jonathan M; Heckerman, David; Graham, Robert R; Plenge, Robert M; Deeks, Steven G; Gianniny, Lauren; Crawford, Gabriel; Sullivan, Jordan; Gonzalez, Elena; Davies, Leela; Camargo, Amy; Moore, Jamie M; Beattie, Nicole; Gupta, Supriya; Crenshaw, Andrew; Burtt, Noël P; Guiducci, Candace; Gupta, Namrata; Gao, Xiaojiang; Qi, Ying; Yuki, Yuko; Piechocka-Trocha, Alicja; Cutrell, Emily; Rosenberg, Rachel; Moss, Kristin L; Lemay, Paul; O'Leary, Jessica; Schaefer, Todd; Verma, Pranshu; Toth, Ildiko; Block, Brian; Baker, Brett; Rothchild, Alissa; Lian, Jeffrey; Proudfoot, Jacqueline; Alvino, Donna Marie L; Vine, Seanna; Addo, Marylyn M; Allen, Todd M; Altfeld, Marcus; Henn, Matthew R; Le Gall, Sylvie; Streeck, Hendrik; Haas, David W; Kuritzkes, Daniel R; Robbins, Gregory K; Shafer, Robert W; Gulick, Roy M; Shikuma, Cecilia M; Haubrich, Richard; Riddler, Sharon; Sax, Paul E; Daar, Eric S; Ribaudo, Heather J; Agan, Brian; Agarwal, Shanu; Ahern, Richard L; Allen, Brady L; Altidor, Sherly; Altschuler, Eric L; Ambardar, Sujata; Anastos, Kathryn; Anderson, Ben; Anderson, Val; Andrady, Ushan; Antoniskis, Diana; Bangsberg, David; Barbaro, Daniel; Barrie, William; Bartczak, J; Barton, Simon; Basden, Patricia; Basgoz, Nesli; Bazner, Suzane; Bellos, Nicholaos C; Benson, Anne M; Berger, Judith; Bernard, Nicole F; Bernard, Annette M; Birch, Christopher; Bodner, Stanley J; Bolan, Robert K; Boudreaux, Emilie T; Bradley, Meg; Braun, James F; Brndjar, Jon E; Brown, Stephen J; Brown, Katherine; Brown, Sheldon T; Burack, Jedidiah; Bush, Larry M; Cafaro, Virginia; Campbell, Omobolaji; Campbell, John; Carlson, Robert H; Carmichael, J Kevin; Casey, Kathleen K; Cavacuiti, Chris; Celestin, Gregory; Chambers, Steven T; Chez, Nancy; Chirch, Lisa M; Cimoch, Paul J; Cohen, Daniel; Cohn, Lillian E; Conway, Brian; Cooper, David A; Cornelson, Brian; Cox, David T; Cristofano, Michael V; Cuchural, George; Czartoski, Julie L; Dahman, Joseph M; Daly, Jennifer S; Davis, Benjamin T; Davis, Kristine; Davod, Sheila M; DeJesus, Edwin; Dietz, Craig A; Dunham, Eleanor; Dunn, Michael E; Ellerin, Todd B; Eron, Joseph J; Fangman, John J W; Farel, Claire E; Ferlazzo, Helen; Fidler, Sarah; Fleenor-Ford, Anita; Frankel, Renee; Freedberg, Kenneth A; French, Neel K; Fuchs, Jonathan D; Fuller, Jon D; Gaberman, Jonna; Gallant, Joel E; Gandhi, Rajesh T; Garcia, Efrain; Garmon, Donald; Gathe, Joseph C; Gaultier, Cyril R; Gebre, Wondwoosen; Gilman, Frank D; Gilson, Ian; Goepfert, Paul A; Gottlieb, Michael S; Goulston, Claudia; Groger, Richard K; Gurley, T Douglas; Haber, Stuart; Hardwicke, Robin; Hardy, W David; Harrigan, P Richard; Hawkins, Trevor N; Heath, Sonya; Hecht, Frederick M; Henry, W Keith; Hladek, Melissa; Hoffman, Robert P; Horton, James M; Hsu, Ricky K; Huhn, Gregory D; Hunt, Peter; Hupert, Mark J; Illeman, Mark L; Jaeger, Hans; Jellinger, Robert M; John, Mina; Johnson, Jennifer A; Johnson, Kristin L; Johnson, Heather; Johnson, Kay; Joly, Jennifer; Jordan, Wilbert C; Kauffman, Carol A; Khanlou, Homayoon; Killian, Robert K; Kim, Arthur Y; Kim, David D; Kinder, Clifford A; Kirchner, Jeffrey T; Kogelman, Laura; Kojic, Erna Milunka; Korthuis, P Todd; Kurisu, Wayne; Kwon, Douglas S; LaMar, Melissa; Lampiris, Harry; Lanzafame, Massimiliano; Lederman, Michael M; Lee, David M; Lee, Jean M L; Lee, Marah J; Lee, Edward T Y; Lemoine, Janice; Levy, Jay A; Llibre, Josep M; Liguori, Michael A; Little, Susan J; Liu, Anne Y; Lopez, Alvaro J; Loutfy, Mono R; Loy, Dawn; Mohammed, Debbie Y; Man, Alan; Mansour, Michael K; Marconi, Vincent C; Markowitz, Martin; Marques, Rui; Martin, Jeffrey N; Martin, Harold L; Mayer, Kenneth Hugh; McElrath, M Juliana; McGhee, Theresa A; McGovern, Barbara H; McGowan, Katherine; McIntyre, Dawn; Mcleod, Gavin X; Menezes, Prema; Mesa, Greg; Metroka, Craig E; Meyer-Olson, Dirk; Miller, Andy O; Montgomery, Kate; Mounzer, Karam C; Nagami, Ellen H; Nagin, Iris; Nahass, Ronald G; Nelson, Margret O; Nielsen, Craig; Norene, David L; O'Connor, David H; Ojikutu, Bisola O; Okulicz, Jason; Oladehin, Olakunle O; Oldfield, Edward C; Olender, Susan A; Ostrowski, Mario; Owen, William F; Pae, Eunice; Parsonnet, Jeffrey; Pavlatos, Andrew M; Perlmutter, Aaron M; Pierce, Michael N; Pincus, Jonathan M; Pisani, Leandro; Price, Lawrence Jay; Proia, Laurie; Prokesch, Richard C; Pujet, Heather Calderon; Ramgopal, Moti; Rathod, Almas; Rausch, Michael; Ravishankar, J; Rhame, Frank S; Richards, Constance Shamuyarira; Richman, Douglas D; Rodes, Berta; Rodriguez, Milagros; Rose, Richard C; Rosenberg, Eric S; Rosenthal, Daniel; Ross, Polly E; Rubin, David S; Rumbaugh, Elease; Saenz, Luis; Salvaggio, Michelle R; Sanchez, William C; Sanjana, Veeraf M; Santiago, Steven; Schmidt, Wolfgang; Schuitemaker, Hanneke; Sestak, Philip M; Shalit, Peter; Shay, William; Shirvani, Vivian N; Silebi, Vanessa I; Sizemore, James M; Skolnik, Paul R; Sokol-Anderson, Marcia; Sosman, James M; Stabile, Paul; Stapleton, Jack T; Starrett, Sheree; Stein, Francine; Stellbrink, Hans-Jurgen; Sterman, F Lisa; Stone, Valerie E; Stone, David R; Tambussi, Giuseppe; Taplitz, Randy A; Tedaldi, Ellen M; Telenti, Amalio; Theisen, William; Torres, Richard; Tosiello, Lorraine; Tremblay, Cecile; Tribble, Marc A; Trinh, Phuong D; Tsao, Alice; Ueda, Peggy; Vaccaro, Anthony; Valadas, Emilia; Vanig, Thanes J; Vecino, Isabel; Vega, Vilma M; Veikley, Wenoah; Wade, Barbara H; Walworth, Charles; Wanidworanun, Chingchai; Ward, Douglas J; Warner, Daniel A; Weber, Robert D; Webster, Duncan; Weis, Steve; Wheeler, David A; White, David J; Wilkins, Ed; Winston, Alan; Wlodaver, Clifford G; van't Wout, Angelique; Wright, David P; Yang, Otto O; Yurdin, David L; Zabukovic, Brandon W; Zachary, Kimon C; Zeeman, Beth; Zhao, Meng

    2010-12-10

    Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection. PMID:21051598

  19. Acetylcholinesterase, a senile plaque component, affects the fibrillogenesis of amyloid-beta-peptides.

    PubMed

    Alvarez, A; Bronfman, F; Pérez, C A; Vicente, M; Garrido, J; Inestrosa, N C

    1995-12-01

    Acetylcholinesterase (AChE) colocalizes with amyloid-beta peptide (A beta) deposits present in the brain of Alzheimer's patients. Recent studies showed that A beta 1-40 can adopt two different conformational states in solution (an amyloidogenic conformer, A beta ac, and a non-amyloidogenic conformer, A beta nac) which have distinct abilities to form amyloid fibrils. We report here that AChE binds A beta nac and accelerates amyloid formation by the same peptide. No such effect was observed with A beta ac, the amyloidogenic conformer, suggesting that AChE acts as a 'pathological chaperone' inducing a conformational transition from A beta nac into A beta ac in vitro.

  20. The major genetic determinants of HIV-1 control affect HLA class I peptide presentation.

    PubMed

    Pereyra, Florencia; Jia, Xiaoming; McLaren, Paul J; Telenti, Amalio; de Bakker, Paul I W; Walker, Bruce D; Ripke, Stephan; Brumme, Chanson J; Pulit, Sara L; Carrington, Mary; Kadie, Carl M; Carlson, Jonathan M; Heckerman, David; Graham, Robert R; Plenge, Robert M; Deeks, Steven G; Gianniny, Lauren; Crawford, Gabriel; Sullivan, Jordan; Gonzalez, Elena; Davies, Leela; Camargo, Amy; Moore, Jamie M; Beattie, Nicole; Gupta, Supriya; Crenshaw, Andrew; Burtt, Noël P; Guiducci, Candace; Gupta, Namrata; Gao, Xiaojiang; Qi, Ying; Yuki, Yuko; Piechocka-Trocha, Alicja; Cutrell, Emily; Rosenberg, Rachel; Moss, Kristin L; Lemay, Paul; O'Leary, Jessica; Schaefer, Todd; Verma, Pranshu; Toth, Ildiko; Block, Brian; Baker, Brett; Rothchild, Alissa; Lian, Jeffrey; Proudfoot, Jacqueline; Alvino, Donna Marie L; Vine, Seanna; Addo, Marylyn M; Allen, Todd M; Altfeld, Marcus; Henn, Matthew R; Le Gall, Sylvie; Streeck, Hendrik; Haas, David W; Kuritzkes, Daniel R; Robbins, Gregory K; Shafer, Robert W; Gulick, Roy M; Shikuma, Cecilia M; Haubrich, Richard; Riddler, Sharon; Sax, Paul E; Daar, Eric S; Ribaudo, Heather J; Agan, Brian; Agarwal, Shanu; Ahern, Richard L; Allen, Brady L; Altidor, Sherly; Altschuler, Eric L; Ambardar, Sujata; Anastos, Kathryn; Anderson, Ben; Anderson, Val; Andrady, Ushan; Antoniskis, Diana; Bangsberg, David; Barbaro, Daniel; Barrie, William; Bartczak, J; Barton, Simon; Basden, Patricia; Basgoz, Nesli; Bazner, Suzane; Bellos, Nicholaos C; Benson, Anne M; Berger, Judith; Bernard, Nicole F; Bernard, Annette M; Birch, Christopher; Bodner, Stanley J; Bolan, Robert K; Boudreaux, Emilie T; Bradley, Meg; Braun, James F; Brndjar, Jon E; Brown, Stephen J; Brown, Katherine; Brown, Sheldon T; Burack, Jedidiah; Bush, Larry M; Cafaro, Virginia; Campbell, Omobolaji; Campbell, John; Carlson, Robert H; Carmichael, J Kevin; Casey, Kathleen K; Cavacuiti, Chris; Celestin, Gregory; Chambers, Steven T; Chez, Nancy; Chirch, Lisa M; Cimoch, Paul J; Cohen, Daniel; Cohn, Lillian E; Conway, Brian; Cooper, David A; Cornelson, Brian; Cox, David T; Cristofano, Michael V; Cuchural, George; Czartoski, Julie L; Dahman, Joseph M; Daly, Jennifer S; Davis, Benjamin T; Davis, Kristine; Davod, Sheila M; DeJesus, Edwin; Dietz, Craig A; Dunham, Eleanor; Dunn, Michael E; Ellerin, Todd B; Eron, Joseph J; Fangman, John J W; Farel, Claire E; Ferlazzo, Helen; Fidler, Sarah; Fleenor-Ford, Anita; Frankel, Renee; Freedberg, Kenneth A; French, Neel K; Fuchs, Jonathan D; Fuller, Jon D; Gaberman, Jonna; Gallant, Joel E; Gandhi, Rajesh T; Garcia, Efrain; Garmon, Donald; Gathe, Joseph C; Gaultier, Cyril R; Gebre, Wondwoosen; Gilman, Frank D; Gilson, Ian; Goepfert, Paul A; Gottlieb, Michael S; Goulston, Claudia; Groger, Richard K; Gurley, T Douglas; Haber, Stuart; Hardwicke, Robin; Hardy, W David; Harrigan, P Richard; Hawkins, Trevor N; Heath, Sonya; Hecht, Frederick M; Henry, W Keith; Hladek, Melissa; Hoffman, Robert P; Horton, James M; Hsu, Ricky K; Huhn, Gregory D; Hunt, Peter; Hupert, Mark J; Illeman, Mark L; Jaeger, Hans; Jellinger, Robert M; John, Mina; Johnson, Jennifer A; Johnson, Kristin L; Johnson, Heather; Johnson, Kay; Joly, Jennifer; Jordan, Wilbert C; Kauffman, Carol A; Khanlou, Homayoon; Killian, Robert K; Kim, Arthur Y; Kim, David D; Kinder, Clifford A; Kirchner, Jeffrey T; Kogelman, Laura; Kojic, Erna Milunka; Korthuis, P Todd; Kurisu, Wayne; Kwon, Douglas S; LaMar, Melissa; Lampiris, Harry; Lanzafame, Massimiliano; Lederman, Michael M; Lee, David M; Lee, Jean M L; Lee, Marah J; Lee, Edward T Y; Lemoine, Janice; Levy, Jay A; Llibre, Josep M; Liguori, Michael A; Little, Susan J; Liu, Anne Y; Lopez, Alvaro J; Loutfy, Mono R; Loy, Dawn; Mohammed, Debbie Y; Man, Alan; Mansour, Michael K; Marconi, Vincent C; Markowitz, Martin; Marques, Rui; Martin, Jeffrey N; Martin, Harold L; Mayer, Kenneth Hugh; McElrath, M Juliana; McGhee, Theresa A; McGovern, Barbara H; McGowan, Katherine; McIntyre, Dawn; Mcleod, Gavin X; Menezes, Prema; Mesa, Greg; Metroka, Craig E; Meyer-Olson, Dirk; Miller, Andy O; Montgomery, Kate; Mounzer, Karam C; Nagami, Ellen H; Nagin, Iris; Nahass, Ronald G; Nelson, Margret O; Nielsen, Craig; Norene, David L; O'Connor, David H; Ojikutu, Bisola O; Okulicz, Jason; Oladehin, Olakunle O; Oldfield, Edward C; Olender, Susan A; Ostrowski, Mario; Owen, William F; Pae, Eunice; Parsonnet, Jeffrey; Pavlatos, Andrew M; Perlmutter, Aaron M; Pierce, Michael N; Pincus, Jonathan M; Pisani, Leandro; Price, Lawrence Jay; Proia, Laurie; Prokesch, Richard C; Pujet, Heather Calderon; Ramgopal, Moti; Rathod, Almas; Rausch, Michael; Ravishankar, J; Rhame, Frank S; Richards, Constance Shamuyarira; Richman, Douglas D; Rodes, Berta; Rodriguez, Milagros; Rose, Richard C; Rosenberg, Eric S; Rosenthal, Daniel; Ross, Polly E; Rubin, David S; Rumbaugh, Elease; Saenz, Luis; Salvaggio, Michelle R; Sanchez, William C; Sanjana, Veeraf M; Santiago, Steven; Schmidt, Wolfgang; Schuitemaker, Hanneke; Sestak, Philip M; Shalit, Peter; Shay, William; Shirvani, Vivian N; Silebi, Vanessa I; Sizemore, James M; Skolnik, Paul R; Sokol-Anderson, Marcia; Sosman, James M; Stabile, Paul; Stapleton, Jack T; Starrett, Sheree; Stein, Francine; Stellbrink, Hans-Jurgen; Sterman, F Lisa; Stone, Valerie E; Stone, David R; Tambussi, Giuseppe; Taplitz, Randy A; Tedaldi, Ellen M; Telenti, Amalio; Theisen, William; Torres, Richard; Tosiello, Lorraine; Tremblay, Cecile; Tribble, Marc A; Trinh, Phuong D; Tsao, Alice; Ueda, Peggy; Vaccaro, Anthony; Valadas, Emilia; Vanig, Thanes J; Vecino, Isabel; Vega, Vilma M; Veikley, Wenoah; Wade, Barbara H; Walworth, Charles; Wanidworanun, Chingchai; Ward, Douglas J; Warner, Daniel A; Weber, Robert D; Webster, Duncan; Weis, Steve; Wheeler, David A; White, David J; Wilkins, Ed; Winston, Alan; Wlodaver, Clifford G; van't Wout, Angelique; Wright, David P; Yang, Otto O; Yurdin, David L; Zabukovic, Brandon W; Zachary, Kimon C; Zeeman, Beth; Zhao, Meng

    2010-12-10

    Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.

  1. Single amino acid mutation in alpha-helical peptide affect second harmonic generation hyperpolarizability

    NASA Astrophysics Data System (ADS)

    Wei, Jing; Wang, Jin-Yun; Zhang, Min-Yi; Chai, Guo-Liang; Lin, Chen-Sheng; Cheng, Wen-Dan

    2013-01-01

    We investigate the effect of side chain on the first-order hyperpolarizability in α-helical polyalanine peptide with the 10th alanine mutation (Acetyl(ala)9X(ala)7NH2). Structures of various substituted peptides are optimized by ONIOM (DFT: AM1) scheme, and then linear and nonlinear optical properties are calculated by SOS//CIS/6-31G∗ method. The polarizability and first-order hyperpolarizability increase obviously only when 'X' represents phenylalanine, tyrosine and tryptophan. We also discuss the origin of nonlinear optical response and determine what caused the increase of first-order hyperpolarizability. Our results strongly suggest that side chains containing benzene, phenol and indole have important contributions to first-order hyperpolarizability.

  2. The Major Genetic Determinants of HIV-1 Control Affect HLA Class I Peptide Presentation

    PubMed Central

    Pereyra, Florencia; Jia, Xiaoming; McLaren, Paul J.; Telenti, Amalio; de Bakker, Paul I.W.; Walker, Bruce D.; Jia, Xiaoming; McLaren, Paul J.; Ripke, Stephan; Brumme, Chanson J.; Pulit, Sara L.; Telenti, Amalio; Carrington, Mary; Kadie, Carl M.; Carlson, Jonathan M.; Heckerman, David; de Bakker, Paul I.W.; Pereyra, Florencia; de Bakker, Paul I.W.; Graham, Robert R.; Plenge, Robert M.; Deeks, Steven G.; Walker, Bruce D.; Gianniny, Lauren; Crawford, Gabriel; Sullivan, Jordan; Gonzalez, Elena; Davies, Leela; Camargo, Amy; Moore, Jamie M.; Beattie, Nicole; Gupta, Supriya; Crenshaw, Andrew; Burtt, Noël P.; Guiducci, Candace; Gupta, Namrata; Carrington, Mary; Gao, Xiaojiang; Qi, Ying; Yuki, Yuko; Pereyra, Florencia; Piechocka-Trocha, Alicja; Cutrell, Emily; Rosenberg, Rachel; Moss, Kristin L.; Lemay, Paul; O’Leary, Jessica; Schaefer, Todd; Verma, Pranshu; Toth, Ildiko; Block, Brian; Baker, Brett; Rothchild, Alissa; Lian, Jeffrey; Proudfoot, Jacqueline; Alvino, Donna Marie L.; Vine, Seanna; Addo, Marylyn M.; Allen, Todd M.; Altfeld, Marcus; Henn, Matthew R.; Le Gall, Sylvie; Streeck, Hendrik; Walker, Bruce D.; Haas, David W.; Kuritzkes, Daniel R.; Robbins, Gregory K.; Shafer, Robert W.; Gulick, Roy M.; Shikuma, Cecilia M.; Haubrich, Richard; Riddler, Sharon; Sax, Paul E.; Daar, Eric S.; Ribaudo, Heather J.; Agan, Brian; Agarwal, Shanu; Ahern, Richard L.; Allen, Brady L.; Altidor, Sherly; Altschuler, Eric L.; Ambardar, Sujata; Anastos, Kathryn; Anderson, Ben; Anderson, Val; Andrady, Ushan; Antoniskis, Diana; Bangsberg, David; Barbaro, Daniel; Barrie, William; Bartczak, J.; Barton, Simon; Basden, Patricia; Basgoz, Nesli; Bazner, Suzane; Bellos, Nicholaos C.; Benson, Anne M.; Berger, Judith; Bernard, Nicole F.; Bernard, Annette M.; Birch, Christopher; Bodner, Stanley J.; Bolan, Robert K.; Boudreaux, Emilie T.; Bradley, Meg; Braun, James F.; Brndjar, Jon E.; Brown, Stephen J.; Brown, Katherine; Brown, Sheldon T.; Burack, Jedidiah; Bush, Larry M.; Cafaro, Virginia; Campbell, Omobolaji; Campbell, John; Carlson, Robert H.; Carmichael, J. Kevin; Casey, Kathleen K.; Cavacuiti, Chris; Celestin, Gregory; Chambers, Steven T.; Chez, Nancy; Chirch, Lisa M.; Cimoch, Paul J.; Cohen, Daniel; Cohn, Lillian E.; Conway, Brian; Cooper, David A.; Cornelson, Brian; Cox, David T.; Cristofano, Michael V.; Cuchural, George; Czartoski, Julie L.; Dahman, Joseph M.; Daly, Jennifer S.; Davis, Benjamin T.; Davis, Kristine; Davod, Sheila M.; Deeks, Steven G.; DeJesus, Edwin; Dietz, Craig A.; Dunham, Eleanor; Dunn, Michael E.; Ellerin, Todd B.; Eron, Joseph J.; Fangman, John J.W.; Farel, Claire E.; Ferlazzo, Helen; Fidler, Sarah; Fleenor-Ford, Anita; Frankel, Renee; Freedberg, Kenneth A.; French, Neel K.; Fuchs, Jonathan D.; Fuller, Jon D.; Gaberman, Jonna; Gallant, Joel E.; Gandhi, Rajesh T.; Garcia, Efrain; Garmon, Donald; Gathe, Joseph C.; Gaultier, Cyril R.; Gebre, Wondwoosen; Gilman, Frank D.; Gilson, Ian; Goepfert, Paul A.; Gottlieb, Michael S.; Goulston, Claudia; Groger, Richard K.; Gurley, T. Douglas; Haber, Stuart; Hardwicke, Robin; Hardy, W. David; Harrigan, P. Richard; Hawkins, Trevor N.; Heath, Sonya; Hecht, Frederick M.; Henry, W. Keith; Hladek, Melissa; Hoffman, Robert P.; Horton, James M.; Hsu, Ricky K.; Huhn, Gregory D.; Hunt, Peter; Hupert, Mark J.; Illeman, Mark L.; Jaeger, Hans; Jellinger, Robert M.; John, Mina; Johnson, Jennifer A.; Johnson, Kristin L.; Johnson, Heather; Johnson, Kay; Joly, Jennifer; Jordan, Wilbert C.; Kauffman, Carol A.; Khanlou, Homayoon; Killian, Robert K.; Kim, Arthur Y.; Kim, David D.; Kinder, Clifford A.; Kirchner, Jeffrey T.; Kogelman, Laura; Kojic, Erna Milunka; Korthuis, P. Todd; Kurisu, Wayne; Kwon, Douglas S.; LaMar, Melissa; Lampiris, Harry; Lanzafame, Massimiliano; Lederman, Michael M.; Lee, David M.; Lee, Jean M.L.; Lee, Marah J.; Lee, Edward T.Y.; Lemoine, Janice; Levy, Jay A.; Llibre, Josep M.; Liguori, Michael A.; Little, Susan J.; Liu, Anne Y.; Lopez, Alvaro J.; Loutfy, Mono R.; Loy, Dawn; Mohammed, Debbie Y.; Man, Alan; Mansour, Michael K.; Marconi, Vincent C.; Markowitz, Martin; Marques, Rui; Martin, Jeffrey N.; Martin, Harold L.; Mayer, Kenneth Hugh; McElrath, M. Juliana; McGhee, Theresa A.; McGovern, Barbara H.; McGowan, Katherine; McIntyre, Dawn; Mcleod, Gavin X.; Menezes, Prema; Mesa, Greg; Metroka, Craig E.; Meyer-Olson, Dirk; Miller, Andy O.; Montgomery, Kate; Mounzer, Karam C.; Nagami, Ellen H.; Nagin, Iris; Nahass, Ronald G.; Nelson, Margret O.; Nielsen, Craig; Norene, David L.; O’Connor, David H.; Ojikutu, Bisola O.; Okulicz, Jason; Oladehin, Olakunle O.; Oldfield, Edward C.; Olender, Susan A.; Ostrowski, Mario; Owen, William F.; Pae, Eunice; Parsonnet, Jeffrey; Pavlatos, Andrew M.; Perlmutter, Aaron M.; Pierce, Michael N.; Pincus, Jonathan M.; Pisani, Leandro; Price, Lawrence Jay; Proia, Laurie; Prokesch, Richard C.; Pujet, Heather Calderon; Ramgopal, Moti; Rathod, Almas; Rausch, Michael; Ravishankar, J.; Rhame, Frank S.; Richards, Constance Shamuyarira; Richman, Douglas D.; Robbins, Gregory K.; Rodes, Berta; Rodriguez, Milagros; Rose, Richard C.; Rosenberg, Eric S.; Rosenthal, Daniel; Ross, Polly E.; Rubin, David S.; Rumbaugh, Elease; Saenz, Luis; Salvaggio, Michelle R.; Sanchez, William C.; Sanjana, Veeraf M.; Santiago, Steven; Schmidt, Wolfgang; Schuitemaker, Hanneke; Sestak, Philip M.; Shalit, Peter; Shay, William; Shirvani, Vivian N.; Silebi, Vanessa I.; Sizemore, James M.; Skolnik, Paul R.; Sokol-Anderson, Marcia; Sosman, James M.; Stabile, Paul; Stapleton, Jack T.; Starrett, Sheree; Stein, Francine; Stellbrink, Hans-Jurgen; Sterman, F. Lisa; Stone, Valerie E.; Stone, David R.; Tambussi, Giuseppe; Taplitz, Randy A.; Tedaldi, Ellen M.; Telenti, Amalio; Theisen, William; Torres, Richard; Tosiello, Lorraine; Tremblay, Cecile; Tribble, Marc A.; Trinh, Phuong D.; Tsao, Alice; Ueda, Peggy; Vaccaro, Anthony; Valadas, Emilia; Vanig, Thanes J.; Vecino, Isabel; Vega, Vilma M.; Veikley, Wenoah; Wade, Barbara H.; Walworth, Charles; Wanidworanun, Chingchai; Ward, Douglas J.; Warner, Daniel A.; Weber, Robert D.; Webster, Duncan; Weis, Steve; Wheeler, David A.; White, David J.; Wilkins, Ed; Winston, Alan; Wlodaver, Clifford G.; Wout, Angelique van’t; Wright, David P.; Yang, Otto O.; Yurdin, David L.; Zabukovic, Brandon W.; Zachary, Kimon C.; Zeeman, Beth; Zhao, Meng

    2011-01-01

    Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA–viral peptide interaction as the major factor modulating durable control of HIV infection. PMID:21051598

  3. Development of an analytical strategy for the identification of potential bioactive peptides generated by in vitro tryptic digestion of fish muscle proteins.

    PubMed

    Capriotti, Anna Laura; Cavaliere, Chiara; Foglia, Patrizia; Piovesana, Susy; Samperi, Roberto; Zenezini Chiozzi, Riccardo; Laganà, Aldo

    2015-01-01

    In the last years, food proteins and peptides are attracting great attention because of the emergence of a new field, that of food-derived bioactive peptides. This paper presents a comparison and evaluation of four different experiments for the identification of sarcoplasmic and myofibrillar fish peptides. This study is aimed at the development of a simple and fast method for the identification of peptides that could arise from fish meat if trypsin was the only digestive enzyme acting on fish meat proteins. In particular, we tested the use of ultrafiltration membranes with a molecular weight cutoff of 3,000 Da. Data analysis has shown that the experiment in which there is neither precipitation nor an ultrafiltration step performed better and allowed the identification of a larger number of peptides and potential antimicrobial peptides (AMPs); this workflow provided 473 and 398 total identified peptides and 44 and 18 AMPs for sarcoplasmic and myofibrillar extracts, respectively. This protocol is found to be faster and more straightforward than the other three tested workflows. The developed strategy could be also useful for other food matrices and could provide information about food quality and safety control.

  4. Conjugation of cell-penetrating peptides to parathyroid hormone affects its structure, potency, and transepithelial permeation.

    PubMed

    Kristensen, Mie; de Groot, Anne Marit; Berthelsen, Jens; Franzyk, Henrik; Sijts, Alice; Nielsen, Hanne Mørck

    2015-03-18

    Delivery of therapeutic peptides and proteins by the use of cell-penetrating peptides (CPPs) as carriers has been suggested as a feasible strategy. The aim of the present study was to investigate the effect of conjugating a series of well-known CPPs to the biologically active part of parathyroid hormone, i.e., PTH(1-34), and to evaluate the effect with regard to secondary structure, potency in Saos-2 cells, immunogenicity, safety, as well as the transepithelial permeation across monolayers by using the Caco-2 cell culture model. Further, co-administration of CPP and PTH(1-34) as an alternative to covalent conjugation was compared with regard to the transepithelial permeation. CPP-conjugated PTH(1-34) fusion peptides were successfully expressed in Escherichia coli and purified from inclusion bodies. No clear correlation between the degree of secondary structure of the CPP-conjugated PTH(1-34) fusion peptides and their potency was found, albeit a general decrease in permeation was observed for both N- and C-terminally CPP-conjugated PTH(1-34) as compared to native PTH(1-34). However, attachment of CPP to the N-terminus significantly increased permeation across Caco-2 cell monolayers as compared to the corresponding C-terminally CPP-conjugated PTH(1-34). In addition, the nonaarginine sequence proved to be the only CPP capable of increasing permeation when conjugated to PTH(1-34) as compared to co-administration of CPP and PTH(1-34). This enhancement effect was, however, associated with an unacceptably low level of cell viability. In conclusion, covalent conjugation of CPPs to PTH(1-34) influenced the secondary structure, potency, and transepithelial permeation efficiency of the resulting conjugate, and hence this approach appears not to be favorable as compared to co-administration when optimizing CPP-mediated permeation of PTH(1-34) across an intestinal epithelium.

  5. Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.

    PubMed

    Meiring, Hugo D; Soethout, Ernst C; Poelen, Martien C M; Mooibroek, Dennis; Hoogerbrugge, Ronald; Timmermans, Hans; Boog, Claire J; Heck, Albert J R; de Jong, Ad P J M; van Els, Cécile A C M

    2006-05-01

    Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.

  6. Purification and identification of adipogenesis inhibitory peptide from black soybean protein hydrolysate.

    PubMed

    Kim, Hyun Jeong; Bae, In Young; Ahn, Chang-Won; Lee, Suyong; Lee, Hyeon Gyu

    2007-11-01

    Adipogenesis inhibitory peptide was isolated and identified from black soybean (Rhynchosia volubilis Lour.) hydrolysate. An adipogenesis inhibitor was purified using consecutive methods including: ultrafiltration (MWCO; 3 and 10kDa), gel filtration chromatography (Superdex Peptide 10/300 GL column), and reverse-phase high-performance liquid chromatography (microBondapak C(18) column). Also, the adipogenesis inhibition effect of the purified peptide was measured by observation of droplet of 3T3-L1 adipocyte by Oil Red O staining in the highest active fraction in each step. The peptide was shown to inhibit the differentiation of the 3T3-L1 pre-adipocyte, which was confirmed by morphological study. The adipogenesis inhibitory peptide was purified 71.43-fold from black soybean hydrolysate throughout a five-step purification procedure. The adipogenesis inhibitor was identified to be a tripeptide, Ile-Gln-Asn, having an IC(50) value of 0.014 mg protein/ml. Furthermore, the synthetic tripeptide (Ile-Gln-Asn) exhibited the similar adipogenesis effects to the purified peptide. Thus, these results showed the potential anti-obesity effect of the purified peptide through control of adiposity.

  7. Identification of SNAIL1 Peptide-Based Irreversible Lysine-Specific Demethylase 1-Selective Inactivators.

    PubMed

    Itoh, Yukihiro; Aihara, Keisuke; Mellini, Paolo; Tojo, Toshifumi; Ota, Yosuke; Tsumoto, Hiroki; Solomon, Viswas Raja; Zhan, Peng; Suzuki, Miki; Ogasawara, Daisuke; Shigenaga, Akira; Inokuma, Tsubasa; Nakagawa, Hidehiko; Miyata, Naoki; Mizukami, Tamio; Otaka, Akira; Suzuki, Takayoshi

    2016-02-25

    Inhibition of lysine-specific demethylase 1 (LSD1), a flavin-dependent histone demethylase, has recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors. This study describes the discovery of SNAIL1 peptide-based inactivators of LSD1. We designed and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane moiety. Among them, peptide 3, bearing hydrazine, displayed the most potent LSD1-inhibitory activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide 3 is a mechanism-based LSD1 inhibitor. Furthermore, peptides 37 and 38, which consist of cell-membrane-permeable oligoarginine conjugated with peptide 3, induced a dose-dependent increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to exhibit LSD1-inhibitory activity intracellularly. In addition, peptide 37 decreased the viability of HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development of potent selective inhibitors and biological probes for LSD1. PMID:26700437

  8. Identification of nodule-specific cysteine-rich plant peptides in endosymbiotic bacteria.

    PubMed

    Durgo, Hajnalka; Klement, Eva; Hunyadi-Gulyas, Eva; Szucs, Attila; Kereszt, Attila; Medzihradszky, Katalin F; Kondorosi, Eva

    2015-07-01

    The symbiosis of Medicago truncatula with Sinorhizobium meliloti or Sinorhizobium medicae soil bacteria results in the formation of root nodules where bacteria inside the plant cells are irreversibly converted to polyploid, nondividing nitrogen-fixing bacteroids. Bacteroid differentiation is host-controlled and the plant effectors are symbiosis-specific secreted plant peptides. In the M. truncatula genome there are more than 600 symbiotic peptide genes including 500 small genes coding for nodule-specific cysteine-rich (NCR) peptides. While NCR transcripts represent >5% of the nodule transcriptome, the existence of only eight NCR peptides has been demonstrated so far. The predicted NCRs are secreted peptides targeted to the endosymbionts. Correspondingly, all the eight detected peptides were present in the bacteroids. Here, we report on large-scale detection of NCR peptides from nodules and from isolated, semipurified endosymbionts at various stages of their differentiation. In total 138 NCRs were detected in the bacteroids; 38 were cationic while the majority was anionic. The presence of early NCRs in nitrogen-fixing bacteroids indicates their high stability, and their long-term maintenance suggests persisting biological roles in the bacteroids.

  9. Identification, synthesis and characterization of a novel antimicrobial peptide HKPLP derived from Hippocampus kuda Bleeker.

    PubMed

    Sun, Dandan; Wu, Songqing; Jing, Chenfeng; Zhang, Ning; Liang, Dong; Xu, Anlong

    2012-03-01

    A novel gene encoding 55 amino-acid residues has been identified from the brooding pouch cDNA library of Hippocampus kuda Bleeker. The deduced amino-acid sequence is highly homologous to several pleurocidin-like peptides from the winter flounder and comprises a signal peptide, a pro-peptide and a mature peptide. The glycine-rich mature peptide, designated HKPLP, contains 24 amino-acid residues and has been synthesized by solid-phase peptide synthesis. The purified HKPLP exhibits antimicrobial activity against several Gram-positive and Gram-negative bacterial strains at low concentrations (MIC 1.5-7.5 μM). Thermal stability assay data show good heat stability. CD spectroscopy experiments indicate that the dominant contents are anti-parallel and parallel sheets, which may have β-sheet or β-strand motif. It is inferred that HKPLP participates in the host defense during egg fertilization and embryo development as an antimicrobial peptide in brooding pouch.

  10. MapReduce Implementation of a Hybrid Spectral Library-Database Search Method for Large-Scale Peptide Identification

    SciTech Connect

    Kalyanaraman, Anantharaman; Cannon, William R.; Latt, Benjamin K.; Baxter, Douglas J.

    2011-11-01

    A MapReduce-based implementation called MR- MSPolygraph for parallelizing peptide identification from mass spectrometry data is presented. The underlying serial method, MSPolygraph, uses a novel hybrid approach to match an experimental spectrum against a combination of a protein sequence database and a spectral library. Our MapReduce implementation can run on any Hadoop cluster environment. Experimental results demonstrate that, relative to the serial version, MR-MSPolygraph reduces the time to solution from weeks to hours, for processing tens of thousands of experimental spectra. Speedup and other related performance studies are also reported on a 400-core Hadoop cluster using spectral datasets from environmental microbial communities as inputs.

  11. Identification of novel peptides for horse meat speciation in highly processed foodstuffs.

    PubMed

    Claydon, Amy J; Grundy, Helen H; Charlton, Adrian J; Romero, M Rosario

    2015-01-01

    There is a need for robust analytical methods to support enforcement of food labelling legislation. Proteomics is emerging as a complementary methodology to existing tools such as DNA and antibody-based techniques. Here we describe the development of a proteomics strategy for the determination of meat species in highly processed foods. A database of specific peptides for nine relevant animal species was used to enable semi-targeted species determination. This principle was tested for horse meat speciation, and a range of horse-specific peptides were identified as heat stable marker peptides for the detection of low levels of horse meat in mixtures with other species.

  12. Identification of novel peptides for horse meat speciation in highly processed foodstuffs.

    PubMed

    Claydon, Amy J; Grundy, Helen H; Charlton, Adrian J; Romero, M Rosario

    2015-01-01

    There is a need for robust analytical methods to support enforcement of food labelling legislation. Proteomics is emerging as a complementary methodology to existing tools such as DNA and antibody-based techniques. Here we describe the development of a proteomics strategy for the determination of meat species in highly processed foods. A database of specific peptides for nine relevant animal species was used to enable semi-targeted species determination. This principle was tested for horse meat speciation, and a range of horse-specific peptides were identified as heat stable marker peptides for the detection of low levels of horse meat in mixtures with other species. PMID:26258799

  13. Serotonin and insulin-like peptides modulate leucokinin-producing neurons that affect feeding and water homeostasis in Drosophila.

    PubMed

    Liu, Yiting; Luo, Jiangnan; Carlsson, Mikael A; Nässel, Dick R

    2015-08-15

    Metabolic homeostasis and water balance is maintained by tight hormonal and neuronal regulation. In Drosophila, insulin-like peptides (DILPs) are key regulators of metabolism, and the neuropeptide leucokinin (LK) is a diuretic hormone that also modulates feeding. However, it is not known whether LK and DILPs act together to regulate feeding and water homeostasis. Because LK neurons express the insulin receptor (dInR), we tested functional links between DILP and LK signaling in feeding and water balance. Thus, we performed constitutive and conditional manipulations of activity in LK neurons and insulin-producing cells (IPCs) in adult flies and monitored food intake, responses to desiccation, and peptide expression levels. We also measured in vivo changes in LK and DILP levels in neurons in response to desiccation and drinking. Our data show that activated LK cells stimulate diuresis in vivo, and that LK and IPC signaling affect food intake in opposite directions. Overexpression of the dInR in LK neurons decreases the LK peptide levels, but only caused a subtle decrease in feeding, and had no effect on water balance. Next we demonstrated that LK neurons express the serotonin receptor 5-HT1B . Knockdown of this receptor in LK neurons diminished LK expression, increased desiccation resistance, and diminished food intake. Live calcium imaging indicates that serotonin inhibits spontaneous activity in abdominal LK neurons. Our results suggest that serotonin via 5-HT1B diminishes activity in the LK neurons and thereby modulates functions regulated by LK peptide, but the action of the dInR in these neurons remains less clear.

  14. HLA-restricted epitope identification and detection of functional T cell responses by using MHC–peptide and costimulatory microarrays

    PubMed Central

    Stone, Jennifer D.; Demkowicz, Walter E.; Stern, Lawrence J.

    2005-01-01

    Identification of T cell epitopes is a vital but often slow and difficult step in studying the immune response to infectious agents and autoantigens. We report a spatially addressable technique for screening large numbers of T cell epitopes for both specific antigen recognition and functional activity induced. This system uses microarrays of immobilized, recombinant MHC–peptide complexes, costimulatory molecules, and cytokine-capture antibodies. The array elements act as synthetic antigen-presenting cells and specifically elicit T cell responses, including adhesion, secretion of cytokines, and modulation of surface markers. The method allows facile identification of pertinent T cell epitopes in a large number of candidates and simultaneous determination of the functional outcome of the interaction. Using this method, we have characterized the activation of human CD4+ and CD8+ T cells responding to vaccinia, influenza, HIV-1, and Epstein–Barr viruses. PMID:15728728

  15. Duplex DNA-Invading γ-Modified Peptide Nucleic Acids Enable Rapid Identification of Bloodstream Infections in Whole Blood

    PubMed Central

    Nölling, Jörk; Rapireddy, Srinivas; Amburg, Joel I.; Crawford, Elizabeth M.; Prakash, Ranjit A.; Rabson, Arthur R.

    2016-01-01

    ABSTRACT Bloodstream infections are a leading cause of morbidity and mortality. Early and targeted antimicrobial intervention is lifesaving, yet current diagnostic approaches fail to provide actionable information within a clinically viable time frame due to their reliance on blood culturing. Here, we present a novel pathogen identification (PID) platform that features the use of duplex DNA-invading γ-modified peptide nucleic acids (γPNAs) for the rapid identification of bacterial and fungal pathogens directly from blood, without culturing. The PID platform provides species-level information in under 2.5 hours while reaching single-CFU-per-milliliter sensitivity across the entire 21-pathogen panel. The clinical utility of the PID platform was demonstrated through assessment of 61 clinical specimens, which showed >95% sensitivity and >90% overall correlation to blood culture findings. This rapid γPNA-based platform promises to improve patient care by enabling the administration of a targeted first-line antimicrobial intervention. PMID:27094328

  16. Purification and identification of antioxidant peptides from walnut (Juglans regia L.) protein hydrolysates.

    PubMed

    Chen, Ning; Yang, Hongmei; Sun, Yi; Niu, Jun; Liu, Shuying

    2012-12-01

    Walnut proteins were hydrolyzed separately using three different proteases to obtain antioxidant peptides. The antioxidant activities of the hydrolysates were measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay. Among hydrolysates, pepsin hydrolysate obtained by 3h exhibited the highest antioxidant activities, which could also quench the hydroxyl radical, chelate ferrous ion, exhibit reducing power and inhibit the lipid peroxidation. Then, 3-h pepsin hydrolysates were purified sequentially by ultrafiltration, gel filtration and RP-HPLC. The sequence of the peptide with the highest antioxidative activity was identified to be Ala-Asp-Ala-Phe (423.23 Da) using RP-HPLC-ESI-MS, which was identified for the first time from walnut protein hydrolysates. Last, the inhibition of the peptide on lipid peroxidation was similar with that of reduced glutathione (GSH). These results indicate that the protein hydrolysates and/or its isolated peptides may be effectively used as food additives.

  17. Identification of pro-opiomelanocortin and secretion of its peptide fragments in bovine adrenals

    SciTech Connect

    Tennov, A.V.; Dmitriev, A.D.; Kizim, E.A.; Ustinova, E.E.

    1986-01-01

    This paper describes the results of an investigation to show that biosynthesis of POMC, its proteolytic processing, an secretion of the peptide products of that processing take place in the bovine adrenals. Rabbit antisera against endorphins were obtained and used for radioimmunoassay of peptides. I 125-labeled peptides were obtained by the chloramine method and purified from free I 125 on Sephadex G-10 (0.7 x 5 cm, centrifugation for 10 min at 1500 g). To detect secretion of peptide fragments of POMC in the adrenals experiments were undertaken to determine the beta-endorphin content in perfusates obtained during retrograde perfusion of the bovine adrenals. It was found that immunoreactive compounds, indistinguishable in their immunochemical properties from beta-endorphin, are present in the perfusates, just as in the tissue extracts.

  18. Identification and characterization of antioxidant peptides obtained by gastrointestinal digestion of amaranth proteins.

    PubMed

    Orsini Delgado, María C; Nardo, Agustina; Pavlovic, Marija; Rogniaux, Hélène; Añón, María C; Tironi, Valeria A

    2016-04-15

    The objective of the present work was to separate and identify antioxidant peptides from a simulated gastrointestinal digest (Id) from Amaranthus mantegazzianus proteins (I), which has previously been demonstrated to have this activity. I and Id were separated by preparative RP-HPLC. Fractions were evaluated by the ORAC method and the more active ones were analyzed by LC-MS/MS. Each fraction presented diverse peptides from different proteins, most of them from the 11S globulin. After grouping the peptides from 11S globulin according to their overlapping sequences, and based on previous information about structure-activity relationships, ten sequences were synthesized, in order to evaluate their antioxidant activity. Four peptides presented interesting activity: AWEEREQGSR>YLAGKPQQEH∼IYIEQGNGITGM∼TEVWDSNEQ. They exhibited some of the structural characteristics already known to demonstrate this activity, all of them containing at least one bulky aromatic residue. All belonged to little structured, internal or exposed regions of the acid subunit of the 11S globulin.

  19. Identification of a putative motif for binding of peptides to HLA-DQ2.

    PubMed

    Johansen, B H; Vartdal, F; Eriksen, J A; Thorsby, E; Sollid, L M

    1996-02-01

    To understand the rules determining peptide binding to the celiac disease and type 1 diabetes mellitus associated HLA-DQ2 molecule, we have studies in detail the binding of a peptide OVA 258-276Y (IINFEKLTEWTSSNVMEERY) which exhibits strong binding to DQ2. First we tested a set of N- and C-terminal truncated variants, and found the core binding region to comprise residues 267-276Y. Single alanine substitution analysis of the OVA 267-276Y peptide revealed that replacements of V272, E275 and the C-terminal Y had negative effects whereas the substitution of N271 had a positive effect. A polyalanine analogue of the OVA 267-276Y peptide with V272, E275 and a C-terminal Y bound at least as well as the original peptide. A variant peptide with a deletion of R276 displayed decreased binding, suggesting that the anchor residues were out of frame in this analogue. To further characterize the residues playing a role in the binding of the OVA 267-276Y peptide to DQ2 we tested the binding of several analogues with substitutions for V272, E275 and the C-terminal Y residue. Our results indicate that peptides binding to DQ2 have anchor residues in relative positions 4, 7 and (P4, P7 and P9). Residues with negatively charged or hydrophobic aliphatic but not positively charged side chains are preferred in P4 and P7, whereas residues with bulky hydrophobic side chains are preferred in P9. PMID:8671602

  20. Affecting proton mobility in activated peptide and whole protein ions via lysine guanidination.

    PubMed

    Pitteri, Sharon J; Reid, Gavin E; McLuckey, Scott A

    2004-01-01

    We have evaluated the effect of lysine guanidination in peptides and proteins on the dissociation of protonated ions in the gas phase. The dissociation of guanidinated model peptide ions compared to their unmodified forms showed behavior consistent with concepts of proton mobility as a major factor in determining favored fragmentation channels. Reduction of proton mobility associated with lysine guanidination was reflected by a relative increase in cleavages occurring C-terminal to aspartic acid residues as well as increases in small molecule losses. To evaluate the effect of guanidination on the dissociation behavior of whole protein ions, bovine ubiquitin was selected as a model. Essentially, all of the amide bond cleavages associated with the +10 charge state of fully guanidinated ubiquitin were observed to occur C-terminal to aspartic acid residues, unlike the dissociation behavior of the +10 ion of the unmodified protein, where competing cleavage N-terminal to proline and nonspecific amide bond cleavages were also observed. The +8 and lower charge states of the guanidinated protein showed prominent losses of small neutral molecules. This overall fragmentation behavior is consistent with current hypotheses regarding whole protein dissociation that consider proton mobility and intramolecular charge solvation as important factors in determining favored dissociation channels, and are also consistent with the fragmentation behaviors observed for the guanidinated model peptide ions. Further evaluation of the utility of condensed phase guanidination of whole proteins is necessary but the results described here confirm that guanidination can be an effective strategy for enhancing C-terminal aspartic acid cleavages. Gas phase dissociation exclusively at aspartic acid residues, especially for whole protein ions, could be useful in identifying and characterizing proteins via tandem mass spectrometry of whole protein ions.

  1. Identification of beryllium-dependent peptides recognized by CD4+ T cells in chronic beryllium disease.

    PubMed

    Falta, Michael T; Pinilla, Clemencia; Mack, Douglas G; Tinega, Alex N; Crawford, Frances; Giulianotti, Marc; Santos, Radleigh; Clayton, Gina M; Wang, Yuxiao; Zhang, Xuewu; Maier, Lisa A; Marrack, Philippa; Kappler, John W; Fontenot, Andrew P

    2013-07-01

    Chronic beryllium disease (CBD) is a granulomatous disorder characterized by an influx of beryllium (Be)-specific CD4⁺ T cells into the lung. The vast majority of these T cells recognize Be in an HLA-DP–restricted manner, and peptide is required for T cell recognition. However, the peptides that stimulate Be-specific T cells are unknown. Using positional scanning libraries and fibroblasts expressing HLA-DP2, the most prevalent HLA-DP molecule linked to disease, we identified mimotopes and endogenous self-peptides that bind to MHCII and Be, forming a complex recognized by pathogenic CD4⁺ T cells in CBD. These peptides possess aspartic and glutamic acid residues at p4 and p7, respectively, that surround the putative Be-binding site and cooperate with HLA-DP2 in Be coordination. Endogenous plexin A peptides and proteins, which share the core motif and are expressed in lung, also stimulate these TCRs. Be-loaded HLA-DP2–mimotope and HLA-DP2–plexin A4 tetramers detected high frequencies of CD4⁺ T cells specific for these ligands in all HLADP2+ CBD patients tested. Thus, our findings identify the first ligand for a CD4⁺ T cell involved in metal-induced hypersensitivity and suggest a unique role of these peptides in metal ion coordination and the generation of a common antigen specificity in CBD.

  2. Identification of Shc Src homology 2 domain-binding peptoid-peptide hybrids.

    PubMed

    Choi, Won Jun; Kim, Sung-Eun; Stephen, Andrew G; Weidlich, Iwona; Giubellino, Alessio; Liu, Fa; Worthy, Karen M; Bindu, Lakshman; Fivash, Matthew J; Nicklaus, Marc C; Bottaro, Donald P; Fisher, Robert J; Burke, Terrence R

    2009-03-26

    A fluorescence anisotropy (FA) competition-based Shc Src homology 2 (SH2) domain-binding was established using the high affinity fluorescein isothiocyanate (FITC) containing peptide, FITC-NH-(CH2)4-CO-pY-Q-G-L-S-amide (8; Kd = 0.35 microM). Examination of a series of open-chain bis-alkenylamide containing peptides, prepared as ring-closing metathesis precursors, showed that the highest affinities were obtained by replacement of the original Gly residue with N alpha-substituted Gly (NSG) "peptoid" residues. This provided peptoid-peptide hybrids of the form "Ac-pY-Q-[NSG]-L-amide." Depending on the NSG substituent, certain of these hybrids exhibited up to 40-fold higher Shc SH2 domain-binding affinity than the parent Gly-containing peptide (IC50 = 248 microM) (for example, for N-homoallyl analogue 50, IC50 = 6 microM). To our knowledge, this work represents the first successful example of the application of peptoid-peptide hybrids in the design of SH2 domain-binding antagonists. These results could provide a foundation for further structural optimization of Shc SH2 domain-binding peptide mimetics. PMID:19226165

  3. Identification of ageing-associated naturally occurring peptides in human urine

    PubMed Central

    Nkuipou-Kenfack, Esther; Bhat, Akshay; Klein, Julie; Jankowski, Vera; Mullen, William; Vlahou, Antonia; Dakna, Mohammed; Koeck, Thomas; Schanstra, Joost P.; Zürbig, Petra; Rudolph, Karl L.; Schumacher, Björn; Pich, Andreas; Mischak, Harald

    2015-01-01

    To assess normal and pathological peptidomic changes that may lead to an improved understanding of molecular mechanisms underlying ageing, urinary peptidomes of 1227 healthy and 10333 diseased individuals between 20 and 86 years of age were investigated. The diseases thereby comprised diabetes mellitus, renal and cardiovascular diseases. Using age as a continuous variable, 116 peptides were identified that significantly (p < 0.05; |ρ|≥0.2) correlated with age in the healthy cohort. The same approach was applied to the diseased cohort. Upon comparison of the peptide patterns of the two cohorts 112 common age-correlated peptides were identified. These 112 peptides predominantly originated from collagen, uromodulin and fibrinogen. While most fibrillar and basement membrane collagen fragments showed a decreased age-related excretion, uromodulin, beta-2-microglobulin and fibrinogen fragments showed an increase. Peptide-based in silico protease analysis was performed and 32 proteases, including matrix metalloproteinases and cathepsins, were predicted to be involved in ageing. Identified peptides, predicted proteases and patient information were combined in a systems biology pathway analysis to identify molecular pathways associated with normal and/or pathological ageing. While perturbations in collagen homeostasis, trafficking of toll-like receptors and endosomal pathways were commonly identified, degradation of insulin-like growth factor-binding proteins was uniquely identified in pathological ageing. PMID:26431327

  4. Identification of ageing-associated naturally occurring peptides in human urine.

    PubMed

    Nkuipou-Kenfack, Esther; Bhat, Akshay; Klein, Julie; Jankowski, Vera; Mullen, William; Vlahou, Antonia; Dakna, Mohammed; Koeck, Thomas; Schanstra, Joost P; Zürbig, Petra; Rudolph, Karl L; Schumacher, Björn; Pich, Andreas; Mischak, Harald

    2015-10-27

    To assess normal and pathological peptidomic changes that may lead to an improved understanding of molecular mechanisms underlying ageing, urinarypeptidomes of 1227 healthy and 10333 diseased individuals between 20 and 86 years of age were investigated. The diseases thereby comprised diabetes mellitus, renal and cardiovascular diseases. Using age as a continuous variable, 116 peptides were identified that significantly (p < 0.05; |ρ|≥0.2) correlated with age in the healthy cohort. The same approach was applied to the diseased cohort. Upon comparison of the peptide patterns of the two cohorts 112 common age-correlated peptides were identified. These 112 peptides predominantly originated from collagen, uromodulin and fibrinogen. While most fibrillar and basement membrane collagen fragments showed a decreased age-related excretion, uromodulin, beta-2-microglobulin and fibrinogen fragments showed an increase. Peptide-based in silico protease analysis was performed and 32 proteases, including matrix metalloproteinases and cathepsins, were predicted to be involved in ageing. Identified peptides, predicted proteases and patient information were combined in a systems biology pathway analysis to identify molecular pathways associated with normal and/or pathological ageing. While perturbations in collagen homeostasis, trafficking of toll-like receptors and endosomal pathways were commonly identified, degradation of insulin-like growth factor-binding proteins was uniquely identified in pathological ageing. PMID:26431327

  5. Identification of immunogenic MAGED4B peptides for vaccine development in oral cancer immunotherapy

    PubMed Central

    Lim, Kue Peng; Chun, Nicole Ai Leng; Gan, Chai Phei; Teo, Soo-Hwang; Rahman, Zainal Ariff Abdul; Abraham, Mannil Thomas; Zain, Rosnah Binti; Ponniah, Sathibalan; Cheong, Sok Ching

    2014-01-01

    The ever-increasing number of tumor-associated antigens has provided a major stimulus for the development of therapeutic peptides vaccines. Tumor-associated peptides can induce high immune response rates and have been developed as vaccines for several types of solid tumors, and many are at various stages of clinical testing. MAGED4B, a melanoma antigen, is overexpressed in oral squamous cell carcinoma (OSCC) and this expression promotes proliferation and cell migration. In this study, we have identified 9 short peptides derived from MAGED4B protein that are restricted in binding to the HLA subtypes common in the Asian population (HLA-A2, A11, and A24). The peptides had good binding affinity with the MHC-Class I molecules and stimulated ex-vivo IFN-gamma and Granzyme-B production in blood samples from OSCC patients, suggesting that they are immunogenic. Further, T cells stimulated with peptide-pulsed dendritic cells showed enhanced T-cell cytotoxic activity against MAGED4B-overexpressing OSCC cell lines. In summary, we have identified MAGED4B peptides that induce anti-tumor immune responses advocating that they could be further developed as vaccine candidates for the treatment of OSCC. PMID:25483651

  6. Mass spectrometric identification of a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes

    PubMed Central

    1995-01-01

    We and others have previously reported that melanoma-specific, cytotoxic T lymphocytes (CTL) define a minimum of six class I-presented peptide epitopes common to most HLA-A2+ melanomas. Here we show that three of these peptide epitopes are coordinately recognized by a CTL clone obtained by limiting dilution from the peripheral blood of an HLA- A2+ melanoma patient. Tandem mass spectrometry was used to characterize and sequence one of these three naturally processed melanoma peptides. One of the potential forms of the deduced peptide sequence (XXTVXXGVX, X = I or L) matches positions 32-40 of the recently identified melanoma gene MART-1/Melan-A. This peptide (p939; ILTVILGVL) binds to HLA-A2 with an intermediate-to-low affinity and is capable of sensitizing the HLA-A2+ T2 cell line to lysis by CTL lines and clones derived from five different melanoma patients. A relative high frequency of anti-p939- specific effector cells appear to be present in situ in HLA-A2+ melanoma patients, since p939 is also recognized by freshly isolated tumor infiltrating lymphocytes. p939 represents a good candidate for the development of peptide-based immunotherapies for the treatment of patients with melanoma. PMID:7807017

  7. Antilisterial peptides from Spanish dry-cured hams: Purification and identification.

    PubMed

    Castellano, Patricia; Mora, Leticia; Escudero, Elizabeth; Vignolo, Graciela; Aznar, Rosa; Toldrá, Fidel

    2016-10-01

    The typical Spanish dry-cured ham has a particular sensory quality that makes it a distinctive food, highly appreciated for consumers worldwide. Its particular physicochemical properties, such as high salt content and reduced water activity contribute to their shelf-stability. However, post-processing actions carried out for the commercialization of these products such as slicing may increase the risk of development of pathogenic microorganisms as Listeria monocytogenes. During ripening, muscle proteins are hydrolyzed by muscle peptidases releasing peptides and free amino acids. Some of these peptides have been described to exert biological activities such as antioxidant and ACE-inhibition. In this study, a peptidomic strategy using mass spectrometry techniques has been used to identify and sequence those naturally generated peptides showing antilisterial activity. One hundred and five peptides have been identified in active fractions and some synthesized and their MIC calculated. Ten peptides were able to inhibit the growth of L. monocytogenes, being the pentapeptide RHGYM the most effective showing a MIC value of 6.25 mM. This study proves for the first time the potential antimicrobial action against L. monocytogenes of certain naturally generated peptides obtained from Spanish dry-cured ham. PMID:27375254

  8. Purification and identification of three novel antioxidant peptides from Cornu Bubali (water buffalo horn).

    PubMed

    Liu, Rui; Wang, Min; Duan, Jin-Ao; Guo, Jian-Ming; Tang, Yu-Ping

    2010-05-01

    Cornu Bubali (water buffalo horn, WBH) has been used in Traditional Chinese Medicine (TCM) for thousands of years. In the present study, three peptides with antioxidant properties were purified from aqueous extract of Cornu Bubali (water buffalo horn, WBH) by consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography and high performance liquid chromatography. The sequences of the three peptides were identified to be Gln-Tyr-Asp-Gln-Gly-Val (WBH-1, 708Da), Tyr-Glu-Asp-Cys-Thr-Asp-Cys-Gly-Asn (WBH-2, 1018Da) and Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn (WBH-3, 1271Da) by matrix assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-LIFT-TOF/TOF MS). The antioxidant activity of these peptides was tested using 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay directly. Methylthiazol tetrazolium (MTT) assay and lactate dehydrogenase (LDH) release assay were also used to evaluate the protection of peptides against hydrogen peroxide (H(2)O(2)) induced injury. The results showed that these peptides could reduce the DPPH radical and protect rat cerebral microvascular endothelial cells (rCMECs) against H(2)O(2)-induced injury, thus demonstrating that these peptides had antioxidant activity. These results suggest that WBH-1, WBH-2 and WBH-3, isolated from the aqueous extract of water buffalo horn are natural antioxidants and may contribute to the efficacy of WBH.

  9. Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish.

    PubMed

    Douglas, Susan E; Gallant, Jeffrey W; Liebscher, Ryan S; Dacanay, Andrew; Tsoi, Stephen C M

    2003-01-01

    Antimicrobial peptides play a crucial role as the first line of defense against invading pathogens. Several types of antimicrobial peptides have been isolated from fish, mostly of the cationic alpha-helical variety. Here, we present the cDNA sequences of five highly disulphide-bonded hepcidin-like peptides from winter flounder, Pseudopleuronectes americanus (Walbaum) and two from Atlantic salmon, Salmo salar (L.). These hepcidin-like molecules consist of a 24 amino acid signal peptide and an acidic propiece of 38-40 amino acids in addition to the mature processed peptide of 19-27 amino acids. Exhaustive data mining of GenBank with these sequences revealed that similar peptides are encoded in the genomes of Japanese flounder, rainbow trout, hybrid striped bass and medaka, indicating that they are widespread among fish. Southern hybridization analysis suggests that closely related hepcidin-like genes are present in other flatfish species, and that they exist as a multigene family clustered on the winter flounder genome. Hepcidin variants are differentially expressed during bacterial challenge, during larval development of P. americanus and in different tissues of adult fish.

  10. Identification of antimicrobial peptides and immobilization strategy suitable for a covalent surface coating with biocompatible properties.

    PubMed

    Rapsch, Karsten; Bier, Frank F; Tadros, Monier; von Nickisch-Rosenegk, Markus

    2014-02-19

    Bacterial accumulation on solid material displays a major source of biomaterial associated infections, cross contamination, and spreading. To overcome these problems, different investigations on surface modifications for the containment of bacterial adhesion have been done. The aim of this research is the development of a rapid and efficient screening procedure to identify and investigate biologically active peptides in an immobilized state in order to produce an antimicrobial surface coating. We figured out that the antimicrobial mode of action is the most important parameter because only peptides with pronounced membrane disruption abilities displayed meaningful activity in an immobilized state. In addition, we highlighted the influence of the coupling reaction chemistry on the activity and amount of the immobilized peptide. Thereupon we developed an optimized antimicrobial surface coating with unrestricted antimicrobial properties by adjusting the immobilization strategy in combination with lowering the necessary peptide amount. Moreover we demonstrated that this antimicrobial surface coating displayed no cytotoxic activity against a eukaryotic cell line and thereby indicates a promising biocompatibility. Furthermore, different antimicrobial peptides obtained either by chemical peptide synthesis or by recombinant DNA technology were used in this study and their activities as well as their potential applications were discussed. PMID:24372365

  11. [Application of reversed-phase liquid chromatography-tandem mass spectrometry in the identification of protein and bioactivity peptides from rape bee pollen].

    PubMed

    Guo, Jing; Yan, Jiaze; Guo, Ming; Jin, Yan

    2014-03-01

    Based on the shotgun proteomic method, rape bee pollen protein was prepared with ultrasonic extraction and digested by trypsin, then separated and sequenced by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), followed by protein database searching. After the above analysis, 353 peptides were identified and the molecular biological functions of 239 proteins have been known. The identified molecular biological functions of these proteins mainly included binding activity, enzyme activity, transporter activity, inhibitor activity and so on. Five peptides were obtained after the screening and appropriate amino acid modification among the identified 353 peptides, according to the relationship between the sequence structure of the bioactivity peptide and angiotensin converting enzyme (ACE) inhibitor activity. The five peptides were speculated to have ACE inhibitor activity and synthesized to detect ACE inhibitor activity. The results showed that all of the five peptides had good ACE inhibitor activity. The peptides of AELDIVLALF and LAVNLIPFP among the five peptides displayed especially good ACE inhibition with half maximal inhibitory concentration (IC50) of (10.65 +/- 0.50) micromol/L and (23.66 +/- 1.08) micromol/L, respectively. This method is rapid, low-cost and achieves the goal of high-throughput screening of bioactivity peptides that greatly shorten the period of identification compared with traditional methods.

  12. Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's Aβ peptide.

    PubMed

    Garvey, Megan; Tepper, Katharina; Haupt, Caroline; Knüpfer, Uwe; Klement, Karolin; Meinhardt, Jessica; Horn, Uwe; Balbach, Jochen; Fändrich, Marcus

    2011-06-10

    The oligomerization of Aβ peptide into amyloid fibrils is a hallmark of Alzheimer's disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aβ and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aβ peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aβ fibrillation. The three histidine residues at positions 6, 13 and 14 of Aβ(1-40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.

  13. Species Identification of Bovine, Ovine and Porcine Type 1 Collagen; Comparing Peptide Mass Fingerprinting and LC-Based Proteomics Methods

    PubMed Central

    Buckley, Mike

    2016-01-01

    Collagen is one of the most ubiquitous proteins in the animal kingdom and the dominant protein in extracellular tissues such as bone, skin and other connective tissues in which it acts primarily as a supporting scaffold. It has been widely investigated scientifically, not only as a biomedical material for regenerative medicine, but also for its role as a food source for both humans and livestock. Due to the long-term stability of collagen, as well as its abundance in bone, it has been proposed as a source of biomarkers for species identification not only for heat- and pressure-rendered animal feed but also in ancient archaeological and palaeontological specimens, typically carried out by peptide mass fingerprinting (PMF) as well as in-depth liquid chromatography (LC)-based tandem mass spectrometric methods. Through the analysis of the three most common domesticates species, cow, sheep, and pig, this research investigates the advantages of each approach over the other, investigating sites of sequence variation with known functional properties of the collagen molecule. Results indicate that the previously identified species biomarkers through PMF analysis are not among the most variable type 1 collagen peptides present in these tissues, the latter of which can be detected by LC-based methods. However, it is clear that the highly repetitive sequence motif of collagen throughout the molecule, combined with the variability of the sites and relative abundance levels of hydroxylation, can result in high scoring false positive peptide matches using these LC-based methods. Additionally, the greater alpha 2(I) chain sequence variation, in comparison to the alpha 1(I) chain, did not appear to be specific to any particular functional properties, implying that intra-chain functional constraints on sequence variation are not as great as inter-chain constraints. However, although some of the most variable peptides were only observed in LC-based methods, until the range of

  14. Identification of Novel Small-Molecule Agonists for Human Formyl Peptide Receptors and Pharmacophore Models of Their RecognitionS⃞

    PubMed Central

    Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Schepetkin, Igor A.; Ye, Richard D.; Rabiet, Marie-Josèphe; Jutila, Mark A.

    2010-01-01

    N-formyl peptide receptor (FPR1) and N-formyl peptide receptor-like 1 (FPRL1, now known as FPR2) are G protein-coupled receptors involved in host defense and sensing cellular dysfunction. Because of the potential for FPR1/FPR2 as a therapeutic target, our recent high-throughput screening efforts have focused on the identification of unique nonpeptide agonists of FPR1/FPR2. In the present studies, we screened a chemolibrary of drug-like molecules for their ability to induce intracellular calcium mobilization in RBL-2H3 cells transfected with human FPR1 or FPR2. Screening of these compounds resulted in the identification of novel and potent agonists that activated both FPR1 and FPR2, as well as compounds that were specific for either FPR1 or FPR2 with EC50 values in the low micromolar range. Specificity of the compounds was supported by analysis of calcium mobilization in HL-60 cells transfected with human FPR1 and FPR2. In addition, all but one agonist activated intracellular calcium flux and chemotaxis in human neutrophils, irrespective of agonist specificity for FPR1 or FPR2. Molecular modeling of the group of FPR1 and FPR2 agonists using field point methodology allowed us to create pharmacophore models for ligand binding sites and formulate requirements for these specific N-formyl peptide receptor agonists. These studies further demonstrate that agonists of FPR1/FPR2 include compounds with wide chemical diversity and that analysis of such compounds can enhance our understanding of their ligand/receptor interaction. PMID:19903830

  15. Purification and Identification of Antioxidant Peptides from Enzymatic Hydrolysate of Spirulina platensis.

    PubMed

    Yu, Jie; Hu, Yuanliang; Xue, Mingxiong; Dun, Yaohao; Li, Shenao; Peng, Nan; Liang, Yunxiang; Zhao, Shumao

    2016-07-28

    The aim of this study was to isolate antioxidant peptides from an enzymatic hydrolysate of Spirulina platensis. A novel antioxidant peptide was obtained by ultrafiltration, gel filtration chromatography, and reverse-phase high-performance liquid chromatography, with the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay used to measure the antioxidant activity, and the sequence was determined to be Pro-Asn-Asn (343.15 Da) by electrospray ionization tandem mass spectrometry. This peptide was synthesized to confirm its antioxidant properties, and it exhibited 81.44 ± 0.43% DPPH scavenging activity at 100 µg/ml, which was similar to that of glutathione (82.63 ± 0.56%). Furthermore, the superoxide anion and hydroxyl free-radical scavenging activities and the SOD activity of the peptide were 47.84 ± 0.49%, 54.01 ± 0.82%, and 12.55 ± 0.75%, respectively, at 10 mg/ml. These results indicate that S. platensis is a good source of antioxidant peptides, and that its hydrolysate may have important applications in the pharmaceutical and food industries. PMID:27090190

  16. Identification of Peptide Mimics of a Glycan Epitope on the Surface of Parasitic Nematode Larvae.

    PubMed

    Umair, Saleh; Deng, Qing; Roberts, Joanna M; Shaw, Richard J; Sutherland, Ian A; Pernthaner, Anton

    2016-01-01

    Phage display was used to identify peptide mimics of an immunologically protective nematode glycan (CarLA) by screening a constrained C7C peptide library for ligands that bound to an anti-CarLA mAb (PAB1). Characterisation of these peptide mimotopes revealed functional similarities with an epitope that is defined by PAB1. Mimotope vaccinations of mice with three selected individual phage clones facilitated the induction of antibody responses that recognised the purified, native CarLA molecule which was obtained from Trichostrongylus colubriformis. Furthermore, these mimotopes are specifically recognised by antibodies in the saliva of animals that were immune to natural polygeneric nematode challenge. This shows that antibodies to the PAB1 epitope form part of the mucosal polyclonal anti-CarLA antibody response of nematode immune host animals. This demonstrates that the selected peptide mimotopes are of biological relevance. These peptides are the first to mimic the PAB1 epitope of CarLA, a defined larval glycan epitope which is conserved between many nematode species. PMID:27579674

  17. Identification of Peptide Mimics of a Glycan Epitope on the Surface of Parasitic Nematode Larvae

    PubMed Central

    Roberts, Joanna M.; Shaw, Richard J.; Sutherland, Ian A.; Pernthaner, Anton

    2016-01-01

    Phage display was used to identify peptide mimics of an immunologically protective nematode glycan (CarLA) by screening a constrained C7C peptide library for ligands that bound to an anti-CarLA mAb (PAB1). Characterisation of these peptide mimotopes revealed functional similarities with an epitope that is defined by PAB1. Mimotope vaccinations of mice with three selected individual phage clones facilitated the induction of antibody responses that recognised the purified, native CarLA molecule which was obtained from Trichostrongylus colubriformis. Furthermore, these mimotopes are specifically recognised by antibodies in the saliva of animals that were immune to natural polygeneric nematode challenge. This shows that antibodies to the PAB1 epitope form part of the mucosal polyclonal anti-CarLA antibody response of nematode immune host animals. This demonstrates that the selected peptide mimotopes are of biological relevance. These peptides are the first to mimic the PAB1 epitope of CarLA, a defined larval glycan epitope which is conserved between many nematode species. PMID:27579674

  18. Identification of a Novel Lysosomal Trafficking Peptide using Phage Display Biopanning Coupled with Endocytic Selection Pressure

    PubMed Central

    2015-01-01

    Methods to select ligands that accumulate specifically in cancer cells and traffic through a defined endocytic pathway may facilitate rapid pairing of ligands with linkers suitable for drug conjugate therapies. We performed phage display biopanning on cancer cells that are treated with selective inhibitors of a given mechanism of endocytosis. Using chlorpromazine to inhibit clathrin-mediated endocytosis in H1299 nonsmall cell lung cancer cells, we identified two clones, ATEPRKQYATPRVFWTDAPG (15.1) and a novel peptide LQWRRDDNVHNFGVWARYRL (H1299.3). The peptides segregate by mechanism of endocytosis and subsequent location of subcellular accumulation. The H1299.3 peptide primarily utilizes clathrin-mediated endocytosis and colocalizes with Lamp1, a lysosomal marker. Conversely, the 15.1 peptide is clathrin-independent and localizes to a perinuclear region. Thus, this novel phage display scheme allows for selection of peptides that selectively internalize into cells via a known mechanism of endocytosis. These types of selections may allow for better matching of linker with targeting ligand by selecting ligands that internalize and traffic to known subcellular locations. PMID:25188559

  19. Identification and Characterization of Peptides That Interact with Hepatitis B Virus via the Putative Receptor Binding Site▿

    PubMed Central

    Deng, Qiang; Zhai, Jian-wei; Michel, Marie-Louise; Zhang, Jun; Qin, Jun; Kong, Yu-ying; Zhang, Xin-xin; Budkowska, Agata; Tiollais, Pierre; Wang, Yuan; Xie, You-hua

    2007-01-01

    A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W1T2X3W4W5) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection. PMID:17192308

  20. Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray.

    PubMed

    Sun, Jiashan; Wang, Xiurong; Wen, Xuexia; Bao, Hongmei; Shi, Lin; Tao, Qimeng; Jiang, Yongping; Zeng, Xianying; Xu, Xiaolong; Tian, Guobin; Zheng, Shimin; Chen, Hualan

    2016-01-01

    Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.

  1. Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

    PubMed Central

    Wen, Xuexia; Bao, Hongmei; Shi, Lin; Tao, Qimeng; Jiang, Yongping; Zeng, Xianying; Xu, Xiaolong; Tian, Guobin; Zheng, Shimin; Chen, Hualan

    2016-01-01

    Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza. PMID:26938453

  2. Solid phase extraction-liquid chromatography (SPE-LC) interface for automated peptide separation and identification by tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Hørning, Ole Bjeld; Theodorsen, Søren; Vorm, Ole; Jensen, Ole Nørregaard

    2007-12-01

    Reversed-phase solid phase extraction (SPE) is a simple and widely used technique for desalting and concentration of peptide and protein samples prior to mass spectrometry analysis. Often, SPE sample preparation is done manually and the samples eluted, dried and reconstituted into 96-well titer plates for subsequent LC-MS/MS analysis. To reduce the number of sample handling stages and increase throughput, we developed a robotic system to interface off-line SPE to LC-ESI-MS/MS. Samples were manually loaded onto disposable SPE tips that subsequently were connected in-line with a capillary chromatography column. Peptides were recovered from the SPE column and separated on the RP-LC column using isocratic elution conditions and analysed by electrospray tandem mass spectrometry. Peptide mixtures eluted within approximately 5 min, with individual peptide peak resolution of ~7 s (FWHM), making the SPE-LC suited for analysis of medium complex samples (3-12 protein components). For optimum performance, the isocratic flow rate was reduced to 30 nL/min, producing nanoelectrospray like conditions which ensure high ionisation efficiency and sensitivity. Using a modified autosampler for mounting and disposing of the SPE tips, the SPE-LC-MS/MS system could analyse six samples per hour, and up to 192 SPE tips in one batch. The relatively high sample throughput, medium separation power and high sensitivity makes the automated SPE-LC-MS/MS setup attractive for proteomics experiments as demonstrated by the identification of the components of simple protein mixtures and of proteins recovered from 2DE gels.

  3. Identification of a Novel Parallel β‐Strand Conformation within Molecular Monolayer of Amyloid Peptide

    PubMed Central

    Liu, Lei; Li, Qiang; Zhang, Shuai; Wang, Xiaofeng; Hoffmann, Søren Vrønning; Li, Jingyuan; Liu, Zheng

    2016-01-01

    The differentiation of protein properties and biological functions arises from the variation in the primary and secondary structure. Specifically, in abnormal assemblies of protein, such as amyloid peptide, the secondary structure is closely correlated with the stable ensemble and the cytotoxicity. In this work, the early Aβ33‐42 aggregates forming the molecular monolayer at hydrophobic interface are investigated. The molecular monolayer of amyloid peptide Aβ33‐42 consisting of novel parallel β‐strand‐like structure is further revealed by means of a quantitative nanomechanical spectroscopy technique with force controlled in pico‐Newton range, combining with molecular dynamic simulation. The identified parallel β‐strand‐like structure of molecular monolayer is distinct from the antiparallel β‐strand structure of Aβ33‐42 amyloid fibril. This finding enriches the molecular structures of amyloid peptide aggregation, which could be closely related to the pathogenesis of amyloid disease.

  4. Identification of IgE binding to Api g 1-derived peptides.

    PubMed

    Ruppel, Elvira; Aÿ, Bernhard; Boisguerin, Prisca; Dölle, Sabine; Worm, Margitta; Volkmer, Rudolf

    2010-11-01

    Celery is a frequent cause of food allergy in pollen-sensitized patients and can induce severe allergic reactions. Clinical symptoms cannot be predicted by skin prick tests (SPTs) or by determining allergen-specific immunoglobulin E (IgE). Our aim was to identify specific IgE binding peptides by using an array technique. For our study, the sera of 21 patients with positive double-blind, placebo-controlled food challenge (DBPCFC) to celery, as well as the sera of 17 healthy patients were used. Additionally, all patients underwent skin tests along with determinations of specific IgE binding. The major allergen of celery Api g 1.0101 (Apium graveolens) was synthesized as an array of overlapping peptides and probed with the patients' sera. We developed an improved immunoassay protocol by investigating peptide lengths, peptide densities, incubation parameters, and readout systems, which could influence IgE binding. Sera of celery-allergic patients showed binding to three distinct regions of Api g 1.0101. The region including amino acids 100 to 126 of Api g 1.0101 is the most important region for IgE binding. This region caused a fivefold higher binding of IgE from the sera of celery-allergic patients compared to those of healthy individuals. In particular, one peptide (VLVPTADGGSIC) was recognized by all sera of celery-allergic patients. In contrast, no binding to this peptide was detected in sera of the healthy controls. Our improved assay strategy allows us to distinguish between celery-allergic and healthy individuals, but needs to be explored in a larger cohort of well-defined patients.

  5. Isolation and identification of a new thymic peptide from calf thymus.

    PubMed

    Qin, Xi-Ming; Duan, Ming-Xing; Deng, Bin; Liu, Xi-Cheng; Zhang, Qiang-Zhe; Liu, Zheng; He, Hong-Xuan

    2004-08-01

    Various thymic peptides (including thymulin, thymic humoral factor, thymopoietin, etc.) play important roles in the process of T cell maturation and development. We isolated a new peptide from calf thymus and named it thymus activity factor II (TAF-II). A yield of 0.92 mg of TAF-II was purified from 500 g calf thymus. Analysis by LC/MSD-Trap showed the amino acid sequence of this hexapeptide to be Glu-Ala-Lys-Ser-Gln-Gly-OH with molecular weight 618.5 daltons. We have also begun to investigate the influence of TAF-II.

  6. Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes.

    PubMed

    Kao, Athit; Chiu, Chi-li; Vellucci, Danielle; Yang, Yingying; Patel, Vishal R; Guan, Shenheng; Randall, Arlo; Baldi, Pierre; Rychnovsky, Scott D; Huang, Lan

    2011-01-01

    Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.

  7. Identification of putative insulin-like peptides and components of insulin signaling pathways in parasitic platyhelminths by the use of genome-wide screening.

    PubMed

    Wang, Shuai; Luo, Xuenong; Zhang, Shaohua; Yin, Cai; Dou, Yongxi; Cai, Xuepeng

    2014-02-01

    No endogenous insulin-like peptides in parasitic flatworms have been reported. Insulin receptors from flukes and tapeworms have been shown to interact directly with the host-derived insulin molecule, which suggests the exploitation of host-derived insulin. In this study, a strategy of genome-wide searches followed by comprehensive analyses of strictly conserved features of the insulin family was used to demonstrate the presence of putative insulin-like peptides in the genomes of six tapeworms and two flukes. In addition, whole insulin signaling pathways were annotated on a genome-wide scale. Two putative insulin-like peptide genes in each genome of tapeworms and one insulin-like peptide gene in each genome of flukes were identified. The comprehensive analyses revealed that all of these peptides showed the common features shared by other members of the insulin family, and the phylogenetic analysis implied a putative gene duplication event in the Cestoda during the evolution of insulin-like peptide genes. The quantitative expression analysis and immunolocalization results suggested a putative role of these peptides in reproduction. Entire sets of major components of the classic insulin signaling pathway were successfully identified, suggesting that this pathway in parasitic flatworms might also regulate many other important biological activities. We believe that the identification of the insulin-like peptides gives us a better understanding of the insulin signaling pathway in these parasites, as well as host-parasite interactions.

  8. Identification of Miscellaneous Peptides from the Skin Secretion of the European Edible Frog, Pelophylax kl. Esculentus.

    PubMed

    Chen, Xiaole; Wang, He; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

    2016-08-01

    The chemical compounds synthesised and secreted from the dermal glands of amphibian have diverse bioactivities that play key roles in the hosts' innate immune system and in causing diverse pharmacological effects in predators that may ingest the defensive skin secretions. As new biotechnological methods have developed, increasing numbers of novel peptides with novel activities have been discovered from this source of natural compounds. In this study, a number of defensive skin secretion peptide sequences were obtained from the European edible frog, P. kl. esculentus, using a 'shotgun' cloning technique developed previously within our laboratory. Some of these sequences have been previously reported but had either obtained from other species or were isolated using different methods. Two new skin peptides are described here for the first time. Esculentin-2c and Brevinin-2Tbe belong to the Esculentin-2 and Brevinin-2 families, respectively, and both are very similar to their respective analogues but with a few amino acid differences. Further, [Asn-3, Lys-6, Phe-13] 3-14-bombesin isolated previously from the skin of the marsh frog, Rana ridibunda, was identified here in the skin of P. kl. esculentus. Studies such as this can provide a rapid elucidation of peptide and corresponding DNA sequences from unstudied species of frogs and can rapidly provide a basis for related scientific studies such as those involved in systematic or the evolution of a large diverse gene family and usage by biomedical researchers as a source of potential novel drug leads or pharmacological agents. PMID:27402449

  9. Sensitivity of Pseudomonas syringae to Bovine Lactoferrin Hydrolysates and Identification of a Novel Inhibitory Peptide

    PubMed Central

    Kim, Woan-Sub; Kim, Pyeung-Hyeun; Shimazaki, Kei-ichi

    2016-01-01

    The antimicrobial activity of bovine lactoferrin hydrolysates (bLFH) was measured against Pseudomonas strains (P. syringae and P. fluorescens) in vitro. To compare susceptibility to bLFH, minimal inhibitory concentration (MIC) values were determined using chemiluminescence assays and paper disc plate assays. Antimicrobial effect against P. fluorescens was not observed by either assay, suggesting that bLFH did not exhibit antimicrobial activity against P. fluorescens. However, a significant inhibition of P. syringae growth was observed in the presence of bLFH. The addition of bLFH in liquid or solid medium inhibited growth of P. syringae in a dose-dependent manner. Furthermore, a bLFH peptide with antimicrobial activity toward P. syringae was isolated and identified. The N-terminal amino acid sequences of thus obtained antimicrobial bLFH peptides were analyzed by a protein sequencer and were found to be Leu-Arg-Ile-Pro-Ser-Lys-Val-Asp-Ser-Ala and Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met. The latter peptide sequence is known to be characteristic of lactoferricin. Therefore, in the present study, we identified a new antimicrobial peptide against P. syringae, present within the N-terminus and possessing the amino acid sequence of Leu-Arg-Ile-Pro-Ser-Lys-Val-Asp-Ser-Ala.

  10. Screening and identification of a specific peptide for targeting hypoxic hepatoma cells.

    PubMed

    Liu, Yiming; Xia, Xiangwen; Wang, Yong; Li, Xin; Zhou, Guofeng; Liang, Huiming; Feng, Gansheng; Zheng, Chuansheng

    2016-08-01

    The biological behaviors of residual hepatoma cells after transarterial embolization therapy, which exist in a hypoxic or even anaerobic tumor microenvironment, differ from the tumor cells under normoxic conditions. This study aimed to use a phage display peptide library for in vivo and in vitro screening to obtain a peptide which could specifically bind to hypoxic hepatoma cells, allowing further targeted diagnosis and treatment for liver cancer. In this study, hypoxic hepatoma cells HepG2 (targeted cells), and normal liver cells HL-7702 (control cells), were utilized to perform three rounds of in vitro screening using a phage-displayed 7-mer peptide library. In addition, hypoxic HepG2 were subcutaneously injected into nude mice to establish a hepatocarcinoma model, followed by performing three rounds of in vivo screening on the phages identified from the in vitro screening. The products from the screening were further identified using ELISA and immunofluorescence staining on cells and tissues. The results indicated that the P11 positive clone had the highest binding effect with hypoxic hepatoma cells. The sequence of the exogenous insert fragment of P11 positive clone was obtained by sequencing: GSTSFSK. The binding assay indicated that GSTSFSK could specifically bind to hypoxic hepatoma cells and hepatocarcinoma tissues. This 7-mer peptide has the potential to be developed as an useful molecular to the targeting diagnosis and treatment of residual hepatoma cells after transarterial chemoembolization. PMID:27381416

  11. Large Improvements in MS/MS Based Peptide Identification Rates using a Hybrid Analysis

    SciTech Connect

    Cannon, William R.; Rawlins, Mitchell M.; Baxter, Douglas J.; Callister, Stephen J.; Lipton, Mary S.; Bryant, Donald A.

    2011-05-06

    We have developed a hybrid method for identifying peptides from global proteomics studies that significantly increases sensitivity and specificity in matching peptides to tandem mass spectra using database searches. The method increased the number of spectra that can be assigned to a peptide in a global proteomics study by 57-147% at an estimated false discovery rate of 5%, with clear room for even greater improvements. The approach combines the general utility of using consensus model spectra typical of database search methods1-3 with the accuracy of the intensity information contained in spectral libraries4-6. This hybrid approach is made possible by recent developments that elucidated the statistical framework common to both data analysis and statistical thermodynamics, resulting in a chemically inspired approach to incorporating fragment intensity information into both database searches and spectral library searches. We applied this approach to proteomics analysis of Synechococcus sp. PCC 7002, a cyanobacterium that is a model organism for studies of photosynthetic carbon fixation and biofuels development. The increased specificity and sensitivity of this approach allowed us to identify many more peptides involved in the processes important for photoautotrophic growth.

  12. Sensitivity of Pseudomonas syringae to Bovine Lactoferrin Hydrolysates and Identification of a Novel Inhibitory Peptide

    PubMed Central

    Kim, Woan-Sub; Kim, Pyeung-Hyeun; Shimazaki, Kei-ichi

    2016-01-01

    The antimicrobial activity of bovine lactoferrin hydrolysates (bLFH) was measured against Pseudomonas strains (P. syringae and P. fluorescens) in vitro. To compare susceptibility to bLFH, minimal inhibitory concentration (MIC) values were determined using chemiluminescence assays and paper disc plate assays. Antimicrobial effect against P. fluorescens was not observed by either assay, suggesting that bLFH did not exhibit antimicrobial activity against P. fluorescens. However, a significant inhibition of P. syringae growth was observed in the presence of bLFH. The addition of bLFH in liquid or solid medium inhibited growth of P. syringae in a dose-dependent manner. Furthermore, a bLFH peptide with antimicrobial activity toward P. syringae was isolated and identified. The N-terminal amino acid sequences of thus obtained antimicrobial bLFH peptides were analyzed by a protein sequencer and were found to be Leu-Arg-Ile-Pro-Ser-Lys-Val-Asp-Ser-Ala and Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met. The latter peptide sequence is known to be characteristic of lactoferricin. Therefore, in the present study, we identified a new antimicrobial peptide against P. syringae, present within the N-terminus and possessing the amino acid sequence of Leu-Arg-Ile-Pro-Ser-Lys-Val-Asp-Ser-Ala. PMID:27621689

  13. Sensitivity of Pseudomonas syringae to Bovine Lactoferrin Hydrolysates and Identification of a Novel Inhibitory Peptide.

    PubMed

    Kim, Woan-Sub; Kim, Pyeung-Hyeun; Shimazaki, Kei-Ichi

    2016-01-01

    The antimicrobial activity of bovine lactoferrin hydrolysates (bLFH) was measured against Pseudomonas strains (P. syringae and P. fluorescens) in vitro. To compare susceptibility to bLFH, minimal inhibitory concentration (MIC) values were determined using chemiluminescence assays and paper disc plate assays. Antimicrobial effect against P. fluorescens was not observed by either assay, suggesting that bLFH did not exhibit antimicrobial activity against P. fluorescens. However, a significant inhibition of P. syringae growth was observed in the presence of bLFH. The addition of bLFH in liquid or solid medium inhibited growth of P. syringae in a dose-dependent manner. Furthermore, a bLFH peptide with antimicrobial activity toward P. syringae was isolated and identified. The N-terminal amino acid sequences of thus obtained antimicrobial bLFH peptides were analyzed by a protein sequencer and were found to be Leu-Arg-Ile-Pro-Ser-Lys-Val-Asp-Ser-Ala and Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met. The latter peptide sequence is known to be characteristic of lactoferricin. Therefore, in the present study, we identified a new antimicrobial peptide against P. syringae, present within the N-terminus and possessing the amino acid sequence of Leu-Arg-Ile-Pro-Ser-Lys-Val-Asp-Ser-Ala. PMID:27621689

  14. Identification and characterization of antioxidant peptides obtained by gastrointestinal digestion of amaranth proteins.

    PubMed

    Orsini Delgado, María C; Nardo, Agustina; Pavlovic, Marija; Rogniaux, Hélène; Añón, María C; Tironi, Valeria A

    2016-04-15

    The objective of the present work was to separate and identify antioxidant peptides from a simulated gastrointestinal digest (Id) from Amaranthus mantegazzianus proteins (I), which has previously been demonstrated to have this activity. I and Id were separated by preparative RP-HPLC. Fractions were evaluated by the ORAC method and the more active ones were analyzed by LC-MS/MS. Each fraction presented diverse peptides from different proteins, most of them from the 11S globulin. After grouping the peptides from 11S globulin according to their overlapping sequences, and based on previous information about structure-activity relationships, ten sequences were synthesized, in order to evaluate their antioxidant activity. Four peptides presented interesting activity: AWEEREQGSR>YLAGKPQQEH∼IYIEQGNGITGM∼TEVWDSNEQ. They exhibited some of the structural characteristics already known to demonstrate this activity, all of them containing at least one bulky aromatic residue. All belonged to little structured, internal or exposed regions of the acid subunit of the 11S globulin. PMID:26675853

  15. Identification of salt-inducible peptide with putative kinase activity in halophilic bacterium Virgibacillus halodenitrificans.

    PubMed

    Rafiee, Mahmoud-Reza; Sokhansanj, Ashrafaddin; Yoosefi, Mitra; Naghizadeh, Mohammad-Ali

    2007-09-01

    Strain XII, a moderately halophilic bacterium, expressed a peptide in response to saline media. This peptide was designated as salt-inducible factor (Sif-A). The purpose of this study is to describe Sif-A, which might be involved in the osmoresistance mechanism of strain XII. The complete sequence of sif-A was determined using PCR. sif-A codes for a polypeptide of 20.518 kDa. The polypeptide has a putative signal peptide of 27 amino acids (2.667 kDa) preceding the mature protein (17.869 kDa). Motif analysis of the deduced amino acid sequence indicated that there is a p-loop NTPase domain on the C-terminal of the peptide, which might correlate with its function. The sequence of the 16S rRNA gene was analyzed phylogenetically to classify strain XII. This organism was found to have the closest association with Virgibacillus halodenitrificans, which was proven by its phenotypic characteristics. PMID:17964480

  16. Antibody-independent identification of bovine milk-derived peptides in breast-milk.

    PubMed

    Picariello, Gianluca; Addeo, Francesco; Ferranti, Pasquale; Nocerino, Rita; Paparo, Lorella; Passariello, Annalisa; Dallas, David C; Robinson, Randall C; Barile, Daniela; Canani, Roberto Berni

    2016-08-10

    Exclusively breast-fed infants can exhibit clear signs of IgE or non IgE-mediated cow's milk allergy. However, the definite characterization of dietary cow's milk proteins (CMP) that survive the maternal digestive tract to be absorbed into the bloodstream and secreted into breast milk remains missing. Herein, we aimed at assessing possible CMP-derived peptides in breast milk. Using high performance liquid chromatography (HPLC)-high resolution mass spectrometry (MS), we compared the peptide fraction of breast milk from 12 donors, among which 6 drank a cup of milk daily and 6 were on a strict dairy-free diet. We identified two bovine β-lactoglobulin (β-Lg, 2 out 6 samples) and one αs1-casein (1 out 6 samples) fragments in breast milk from mothers receiving a cup of bovine milk daily. These CMP-derived fragments, namely β-Lg (f42-54), (f42-57) and αs1-casein (f180-197), were absent in milk from mothers on dairy-free diet. In contrast, neither intact nor hydrolyzed β-Lg was detected by western blot and competitive ELISA in any breast milk sample. Eight additional bovine milk-derived peptides identified by software-assisted MS were most likely false positive. The results of this study demonstrate that CMP-derived peptides rather than intact CMP may sensitize or elicit allergic responses in the neonate through mother's milk. Immunologically active peptides from the maternal diet could be involved in priming the newborn's immune system, driving a tolerogenic response. PMID:27396729

  17. Identification and Characterization of a Peptide Affinity Reagent for Detection of Noroviruses in Clinical Samples

    PubMed Central

    Rogers, Jennifer D.; Ajami, Nadim J.; Fryszczyn, Bartlomiej G.; Estes, Mary K.; Atmar, Robert L.

    2013-01-01

    Norovirus (NoV) is the most common agent of nonbacterial epidemic gastroenteritis and is estimated to cause 21 million cases of the disease in the United States annually. The antigen enzyme-linked immunosorbent assays (ELISAs) currently available for NoV diagnosis detect only certain strains and are approved for use in the United States only in epidemics where NoV is suspected. There is a clear need for simpler, more rapid, and more reliable diagnostic tools for the detection of NoV. In this study, phage display technology was used to screen a library of phage displaying random 12-mer peptides for those that bind to Norwalk virus virus-like particles (NV VLPs). Three phage clones displaying unique peptides were identified, and both the peptide-displaying phages and the peptides were confirmed to bind specifically to NV VLPs. The peptide displayed on phage clone NV-N-R5-1 was determined to bind to the protruding domain of the VP1 capsid protein. This phage also bound to NV VLPs seeded into NoV-negative stool with a limit of detection of 1.56 ng NV VLP. This value was comparable to monoclonal antibody (MAb) 3912, which is currently used in commercially available assays. Furthermore, the NV-N-R5-1 phage exhibited high specificity by detecting NV only in previously characterized NV-positive stool samples in contrast to no detection in NV-negative stool samples. These data demonstrate that the further development of NV-N-R5-1 phage as a diagnostic reagent is possible and might offer several distinct advantages over antibodies, such as decreases in the time and cost of production and ease of isolating phage against other epidemic strains currently circulating as well as those that are emerging. PMID:23554202

  18. Hydrophilic Linkers and Polar Contacts Affect Aggregation of FG Repeat Peptides

    PubMed Central

    Dölker, Nicole; Zachariae, Ulrich; Grubmüller, Helmut

    2010-01-01

    Abstract Transport of large proteins into the nucleus involves two events, binding of the cargo protein to a transport receptor in the cytoplasm and passage of the cargo-transporter complex through the selective permeability barrier of the nuclear pore complex. The permeability barrier is formed by largely disordered polypeptides, each containing a number of conserved hydrophobic phenylalanine-glycine (FG) sequence motifs, connected by hydrophilic linkers of varying sequence (FG nups). How the motifs interact to form the permeability barrier, however, is not yet known. We have, therefore, carried out molecular dynamics simulations on various model FG repeat peptides to study the aggregation propensity of FG nups and the specific roles of the hydrophobic FG motifs and the hydrophilic linkers. Our simulations show spontaneous aggregation of the model nups into hydrated aggregates, which exhibit structural features assumed to be part of the permeability barrier. Our simulations suggest that short β-sheets are an important structural feature of the aggregates and that Phe residues are sufficiently exposed to allow rapid binding of transport receptors. A surprisingly large influence of the amino acid composition of the hydrophilic linkers on aggregation is seen, as well as a major contribution of hydrogen-bonding patterns. PMID:20513410

  19. Optimization of Search Engines and Postprocessing Approaches to Maximize Peptide and Protein Identification for High-Resolution Mass Data.

    PubMed

    Tu, Chengjian; Sheng, Quanhu; Li, Jun; Ma, Danjun; Shen, Xiaomeng; Wang, Xue; Shyr, Yu; Yi, Zhengping; Qu, Jun

    2015-11-01

    The two key steps for analyzing proteomic data generated by high-resolution MS are database searching and postprocessing. While the two steps are interrelated, studies on their combinatory effects and the optimization of these procedures have not been adequately conducted. Here, we investigated the performance of three popular search engines (SEQUEST, Mascot, and MS Amanda) in conjunction with five filtering approaches, including respective score-based filtering, a group-based approach, local false discovery rate (LFDR), PeptideProphet, and Percolator. A total of eight data sets from various proteomes (e.g., E. coli, yeast, and human) produced by various instruments with high-accuracy survey scan (MS1) and high- or low-accuracy fragment ion scan (MS2) (LTQ-Orbitrap, Orbitrap-Velos, Orbitrap-Elite, Q-Exactive, Orbitrap-Fusion, and Q-TOF) were analyzed. It was found combinations involving Percolator achieved markedly more peptide and protein identifications at the same FDR level than the other 12 combinations for all data sets. Among these, combinations of SEQUEST-Percolator and MS Amanda-Percolator provided slightly better performances for data sets with low-accuracy MS2 (ion trap or IT) and high accuracy MS2 (Orbitrap or TOF), respectively, than did other methods. For approaches without Percolator, SEQUEST-group performs the best for data sets with MS2 produced by collision-induced dissociation (CID) and IT analysis; Mascot-LFDR gives more identifications for data sets generated by higher-energy collisional dissociation (HCD) and analyzed in Orbitrap (HCD-OT) and in Orbitrap Fusion (HCD-IT); MS Amanda-Group excels for the Q-TOF data set and the Orbitrap Velos HCD-OT data set. Therefore, if Percolator was not used, a specific combination should be applied for each type of data set. Moreover, a higher percentage of multiple-peptide proteins and lower variation of protein spectral counts were observed when analyzing technical replicates using Percolator

  20. Identification of bioactive peptides in hypoallergenic infant milk formulas by CE-TOF-MS assisted by semiempirical model of electromigration behavior.

    PubMed

    Català-Clariana, Sergio; Benavente, Fernando; Giménez, Estela; Barbosa, José; Sanz-Nebot, Victoria

    2013-07-01

    Biologically active peptides derived from complex bovine milk protein hydrolysates are of particular interest in food science and nutrition because they have been shown to play different physiological roles, providing benefits in human health. In this study, we used CE-TOF-MS for separation and identification of bioactive peptides in three hypoallergenic infant milk formulas. An appropriate sample cleanup using a citrate buffer with DTT and urea followed by SPE with Sep-Pack® C18 and StrataX™ cartridges allowed the detection of a large number of low molecular mass bioactive peptides. This preliminary identification was solely based on the measured experimental monoisotopic molecular mass values (M(exp)). Later, we evaluated the classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (m(e) vs. q/M(α), α = 1/2 for the classical polymer model) to describe their migration behavior. The assistance of migration prediction proved to be useful to improve reliability of the identification, avoiding misinterpretations and solving some identity conflicts. After revision, the identity of 24, 30, and 38 bioactive peptides was confirmed in each of the three infant milk formulas. A significant number of these peptides were reported as inhibitors of angiotensin-converting enzyme, however, the presence of sequences with other biological activities such as antihypertensive, antithrombotic, hypocholesterolemic, immunomodulation, cytotoxicity, antioxidant, antimicrobial, antigenic, or opioid was also confirmed.

  1. Identification of bioactive peptides in a functional yogurt by micro liquid chromatography time-of-flight mass spectrometry assisted by retention time prediction.

    PubMed

    Kunda, Pradeep B; Benavente, Fernando; Catalá-Clariana, Sergio; Giménez, Estela; Barbosa, José; Sanz-Nebot, Victoria

    2012-03-16

    In this study we used micro liquid chromatography coupled to time-of-flight mass spectrometry (microLC-TOF-MS) for separation and identification of bioactive peptides in a yogurt marketed as an antihypertensive functional food. An appropriate sample clean-up using solid-phase extraction (SPE) allowed detection of a large number of low-molecular-mass bioactive peptides by reversed-phase microLC-TOF-MS. The preliminary identification was solely based on the experimental monoisotopic molecular mass values (M(exp)). Later, we evaluated the correlations between predicted normalized elution time (NET) and experimental normalized retention times (t(r)') values to describe the retention behavior of the proposed sequences. The assistance of retention prediction proved to be useful to improve reliability of the identification, avoiding misinterpretations and solving some identity conflicts. After revision, the identity of only fifty bioactive peptides was confirmed. Significant number of these peptides was reported as angiotensin converting enzyme (ACE) inhibitors and nine of them were antihypertensive. The presence of peptide sequences with other biological activities such as antibacterial, antithrombotic, antioxidant, cell modulation, immune or phagocytosis stimulation, epitopes of B cells and opioid agonists was also confirmed.

  2. How the amyloid-β peptide and membranes affect each other: an extensive simulation study.

    PubMed

    Poojari, Chetan; Kukol, Andreas; Strodel, Birgit

    2013-02-01

    The etiology of Alzheimer's disease is thought to be linked to interactions between amyloid-β (Aβ) and neural cell membranes, causing membrane disruption and increased ion conductance. The effects of Aβ on lipid behavior have been characterized experimentally, but structural and causal details are lacking. We used atomistic molecular dynamics simulations totaling over 6 μs in simulation time to investigate the behavior of Aβ(42) in zwitterionic and anionic lipid bilayers. We simulated transmembrane β-sheets (monomer and tetramer) resulting from a global optimization study and a helical structure obtained from an NMR study. In all simulations Aβ(42) remained embedded in the bilayer. It was found that the surface charge and the lipid tail type are determinants for transmembrane stability of Aβ(42) with zwitterionic surfaces and unsaturated lipids promoting stability. From the considered structures, the β-sheet tetramer is most stable as a result of interpeptide interactions. We performed an in-depth analysis of the translocation of water in the Aβ(42)-bilayer systems. We observed that this process is generally fast (within a few nanoseconds) yet generally slower than in the peptide-free bilayers. It is mainly governed by the lipid type, simulation temperature and Aβ(42) conformation. The rate limiting step is the permeation through the hydrophobic core, where interactions between Aβ(42) and permeating H(2)O molecules slow the translocation process. The β-sheet tetramer allows more water molecules to pass through the bilayer compared to monomeric Aβ, allowing us to conclude that the experimentally observed permeabilization of membranes must be due to membrane-bound Aβ oligomers, and not monomers.

  3. Assessment of impact of peptide nucleic acid fluorescence in situ hybridization for rapid identification of coagulase-negative staphylococci in the absence of antimicrobial stewardship intervention.

    PubMed

    Holtzman, Carol; Whitney, Dana; Barlam, Tamar; Miller, Nancy S

    2011-04-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) was instituted at Boston Medical Center for the rapid identification of coagulase-negative staphylococci (CoNS). Without active notification or antimicrobial stewardship intervention, a pre- and postimpact analysis showed no benefit of this assay with respect to the length of hospital stay or vancomycin use.

  4. DNA Subtraction of In Vivo Selected Phage Repertoires for Efficient Peptide Pathology Biomarker Identification in Neuroinflammation Multiple Sclerosis Model

    PubMed Central

    Vargas-Sanchez, Karina; Vekris, Antonios; Petry, Klaus G.

    2016-01-01

    To streamline in vivo biomarker discovery, we developed a suppression subtractive DNA hybridization technique adapted for phage-displayed combinatorial libraries of 12 amino acid peptides (PhiSSH). Physical DNA subtraction is performed in a one-tube-all-reactions format by sequential addition of reagents, producing the enrichment of specific clones of one repertoire. High-complexity phage repertoires produced by in vivo selections in the multiple sclerosis rat model (experimental autoimmune encephalomyelitis, EAE) and matched healthy control rats were used to evaluate the technique. The healthy repertoire served as a physical DNA subtractor from the EAE repertoire to produce the subtraction repertoire. Full next-generation sequencing (NGS) of the three repertoires was performed to evaluate the efficiency of the subtraction technique. More than 96% of the clones common to the EAE and healthy repertoires were absent from the subtraction repertoire, increasing the probability of randomly selecting various specific peptides for EAE pathology to about 70%. Histopathology experiments were performed to confirm the quality of the subtraction repertoire clones, producing distinct labeling of the blood–brain barrier (BBB) affected by inflammation among healthy nervous tissue or the preferential binding to IL1-challenged vs. resting human BBB model. Combining PhiSSH with NGS will be useful for controlled in vivo screening of small peptide combinatorial libraries to discover biomarkers of specific molecular alterations interspersed within healthy tissues. PMID:26917946

  5. Enteric YaiW Is a Surface-Exposed Outer Membrane Lipoprotein That Affects Sensitivity to an Antimicrobial Peptide

    PubMed Central

    Arnold, Markus F. F.; Caro-Hernandez, Paola; Tan, Karen; Runti, Giulia; Wehmeier, Silvia; Scocchi, Marco; Doerrler, William T.; Ferguson, Gail P.

    2014-01-01

    yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions. PMID:24214946

  6. Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

    PubMed Central

    Pinzón, Carlos Giovanni; Curtidor, Hernando; Reyes, Claudia; Méndez, David; Patarroyo, Manuel Elkin

    2008-01-01

    The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite–erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having α-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 μM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion. PMID:18593818

  7. Identification of human adenovirus early region 1 products by using antisera against synthetic peptides corresponding to the predicted carboxy termini.

    PubMed Central

    Yee, S P; Rowe, D T; Tremblay, M L; McDermott, M; Branton, P E

    1983-01-01

    Synthetic peptides were prepared which corresponded to the carboxy termini of the human adenovirus type 5 early region 1B (E1B) 58,000-molecular-weight (58K) protein (Tyr-Ser-Asp-Glu-Asp-Thr-Asp) and of the E1A gene products (Tyr-Gly-Lys-Arg-Pro-Arg-Pro). Antisera raised against these peptides precipitated polypeptides from adenovirus type 5-infected KB cells; serum raised against the 58K carboxy terminus was active against the E1B 58K phosphoprotein, whereas serum raised against the E1A peptide immunoprecipitated four major and at least two minor polypeptides. These latter proteins migrated with apparent molecular weights of 52K, 50K, 48.5K, 45K, 37.5K, and 35K, and all were phosphoproteins. By using tryptic phosphopeptide analysis, the four major species (52K, 50K, 48.5K, and 45K) were found to be related, as would be expected if all were products of the E1A region. The ability of the antipeptide sera to precipitate these viral proteins thus confirmed that the previously proposed sequence of E1 DNA and mRNA and the reading frame of the mRNA are correct. Immunofluorescent-antibody staining with the antipeptide sera indicated that the 58K E1B protein was localized both in the nucleus and in the cytoplasm, especially in the perinuclear region. The E1A-specific serum also stained both discrete patches in the nucleus and diffuse areas of the cytoplasm. These data suggest that both the 58K protein and the E1A proteins may function in or around the nucleus. These highly specific antipeptide sera should allow for a more complete identification and characterization of these important viral proteins. Images PMID:6343626

  8. Identification and Quantitation of Newly Synthesized Proteins in Escherichia coli by Enrichment of Azidohomoalanine-labeled Peptides with Diagonal Chromatography

    PubMed Central

    Kramer, Gertjan; Sprenger, Richard R.; Back, JaapWillem; Dekker, Henk L.; Nessen, Merel A.; van Maarseveen, Jan H.; de Koning, Leo J.; Hellingwerf, Klaas J.; de Jong, Luitzen; de Koster, Chris G.

    2009-01-01

    A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 min and digestion of total protein, azhal-labeled peptides are isolated by a retention time shift between two reversed phase chromatographic runs. The retention time shift is induced by a reaction selective for the azido group in labeled peptides using tris(2-carboxyethyl)phosphine. Selectively modified peptides are identified by reversed phase liquid chromatography and on-line tandem mass spectrometry. We identified 527 proteins representative of all major Gene Ontology categories. Comparing the relative amounts of 344 proteins synthesized in 15 min upon a switch of growth temperature from 37 to 44 °C showed that nearly 20% increased or decreased more than 2-fold. Among the most up-regulated proteins many were chaperones and proteases in accordance with the cells response to unfolded proteins due to heat stress. Comparison of our data with results from previous microarray experiments revealed the importance of regulation of gene expression at the level of transcription of the most elevated proteins under heat shock conditions and enabled identification of several candidate genes whose expression may predominantly be regulated at the level of translation. This work demonstrates for the first time the use of a bioorthogonal amino acid for proteome-wide detection of changes in the amounts of proteins synthesized during a brief period upon variations in cellular growth conditions. Comparison of such data with relative mRNA levels enables assessment of the separate contributions of transcription and translation to the regulation of gene expression. PMID:19321432

  9. Identification and quantitation of newly synthesized proteins in Escherichia coli by enrichment of azidohomoalanine-labeled peptides with diagonal chromatography.

    PubMed

    Kramer, Gertjan; Sprenger, Richard R; Back, JaapWillem; Dekker, Henk L; Nessen, Merel A; van Maarseveen, Jan H; de Koning, Leo J; Hellingwerf, Klaas J; de Jong, Luitzen; de Koster, Chris G

    2009-07-01

    A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 min and digestion of total protein, azhal-labeled peptides are isolated by a retention time shift between two reversed phase chromatographic runs. The retention time shift is induced by a reaction selective for the azido group in labeled peptides using tris(2-carboxyethyl)phosphine. Selectively modified peptides are identified by reversed phase liquid chromatography and on-line tandem mass spectrometry. We identified 527 proteins representative of all major Gene Ontology categories. Comparing the relative amounts of 344 proteins synthesized in 15 min upon a switch of growth temperature from 37 to 44 degrees C showed that nearly 20% increased or decreased more than 2-fold. Among the most up-regulated proteins many were chaperones and proteases in accordance with the cells response to unfolded proteins due to heat stress. Comparison of our data with results from previous microarray experiments revealed the importance of regulation of gene expression at the level of transcription of the most elevated proteins under heat shock conditions and enabled identification of several candidate genes whose expression may predominantly be regulated at the level of translation. This work demonstrates for the first time the use of a bioorthogonal amino acid for proteome-wide detection of changes in the amounts of proteins synthesized during a brief period upon variations in cellular growth conditions. Comparison of such data with relative mRNA levels enables assessment of the separate contributions of transcription and translation to the regulation of gene expression.

  10. Identification and biological activity of ovine and caprine calcitonin receptor-stimulating peptides 1 and 2.

    PubMed

    Charles, Christopher J; Katafuchi, Takeshi; Yandle, Timothy G; Minamino, Naoto

    2008-08-01

    We have recently reported the isolation of three new members of the calcitonin (CT) gene-related peptide family of peptides, the CT receptor (CT-R)-stimulating peptides (CRSPs). We now report the sequencing and characterization of ovine/caprine CRSP-1 and caprine CRSP-2. Mature ovine and caprine CRSP-1 are identical and have strong structural homology to CRSP-1s identified to date from other species. As with other CRSP-1s, ovine/caprine CRSP-1 binds to and activates the CT-R but not the CT-like receptor (CL-R) in combination with the receptor activity-modifying proteins (RAMPs). By contrast, caprine CRSP-2 does not activate any of these receptor-RAMP complexes. Intravenous infusions of ovine CRSP-1 to normal conscious sheep induced dose-dependent reduction in plasma total Ca levels (P=0.02) and corrected Ca levels (P=0.017) associated with increases in plasma cAMP (P=0.002). CRSP-1 reduced both plasma amino-terminal pro-C-type natriuretic peptide levels (P=0.006) and plasma renin activity (P=0.028). There were no significant effects observed on hemodynamic or renal indices measured. In conclusion, we have sequenced ovine/caprine CRSP-1 and caprine CRSP-2 precursors. This newly identified CRSP-1 has been shown to share the structural and biological features of CRSP-1s known to date. In vivo studies confirm that ovine CRSP-1 reduces plasma Ca levels in sheep, presumably via a cAMP-mediated mechanism. By contrast, caprine CRSP-2 did not stimulate any combination of CT-R, CL-R, and RAMPs. Accession numbers of cDNA determined in this study are caprine CRSP-1, AB364646; caprine CRSP-2, AB364647; and ovine CRSP-1, AB364648.

  11. Identification and characterization of Aβ peptide interactors in Alzheimer's disease by structural approaches.

    PubMed

    Philibert, Keith D; Marr, Robert A; Norstrom, Eric M; Glucksman, Marc J

    2014-01-01

    Currently, there are very limited pharmaceutical interventions for Alzheimer's disease (AD) to alleviate the amyloid burden implicated in the pathophysiology of the disease. Alzheimer's disease is characterized immunohistologically by the accumulation of senile plaques in the brain with afflicted patients progressively losing short-term memory and, ultimately, cognition. Although significant improvements in clinical diagnosis and care for AD patients have been made, effective treatments for this devastating disease remain elusive. A key component of the amyloid burden of AD comes from accumulation of the amyloid-beta (Aβ) peptide which comes from processing of the amyloid precursor protein (APP) by enzymes termed secretases, leading to production of these toxic Aβ peptides of 40-42 amino acids. New therapeutic approaches for reducing Aβ are warranted after the most logical avenues of inhibiting secretase activity appear less than optimal in ameliorating the progression of AD.Novel therapeutics may be gleaned from proteomics biomarker initiatives to yield detailed molecular interactions of enzymes and their potential substrates. Explicating the APPome by deciphering protein complexes forming in cells is a complementary approach to unveil novel molecular interactions with the amyloidogenic peptide precursor to both understand the biology and develop potential upstream drug targets. Utilizing these strategies we have identified EC 3.4.24.15 (EP24.15), a zinc metalloprotease related to neprilysin (NEP), with the ability to catabolize Aβ 1-42 by examining first potential in silico docking and then verification by mass spectrometry. In addition, a hormone carrier protein, transthyreitin (TTR), was identified and with its abundance in cerebrospinal fluid (CSF), found to clear Aβ by inhibiting formation of oligomeric forms of Aβ peptide. The confluence of complementary strategies may allow new therapeutic avenues as well as biomarkers for AD that will aid in

  12. Molecular identification and functional characteristics of peptide transporters in the bonnethead shark (Sphyrna tiburo).

    PubMed

    Hart, Hannah R; Evans, Andrew N; Gelsleichter, James; Ahearn, Gregory A

    2016-10-01

    Elasmobranchs are considered to be top marine predators, and in general play important roles in the transfer of energy within marine ecosystems. Despite this, little is known regarding the physiological processes of digestion and nutrient absorption in these fishes. One topic that is particularly understudied is the process of nutrient uptake across the elasmobranch gastrointestinal tract. Given their carnivorous diet, the present study sought to expand knowledge on dietary nutrient uptake in elasmobranchs by focusing on the uptake of products of protein digestion. To accomplish this, a full-length cDNA encoding peptide transporter 1 (PepT1), a protein previously identified within the brush border membrane of vertebrates that is responsible for the translocation of peptides released during digestion by luminal and membrane-bound proteases, was isolated from the bonnethead shark (Sphyrna tiburo). A cDNA encoding the related peptide transporter PepT2 was also isolated from S. tiburo using the same methodology. The presence of PepT1 was then localized in multiple components of the bonnethead digestive tract (esophagus, stomach, duodenum, intestine, rectum, and pancreas) using immunohistochemistry. Vesicle studies were used to identify the apparent affinity of PepT1 and to quantify the rate of dipeptide uptake by its H(+)-dependent cotransporter properties. The results of this study provide insight into the properties of peptide uptake within the bonnethead gut, and can facilitate future work on physiological regulation of protein metabolism and absorption including how these processes may vary in elasmobranchs that exhibit different feeding strategies. PMID:27188191

  13. Proteogenomic strategies for identification of aberrant cancer peptides using large-scale Next Generation Sequencing data

    SciTech Connect

    Woo, Sunghee; Cha, Seong Won; Na, Seungjin; Guest, Clark; Liu, Tao; Smith, Richard D.; Rodland, Karin D.; Payne, Samuel H.; Bafna, Vineet

    2014-11-17

    Cancer is driven by the acquisition of somatic DNA lesions. Distinguishing the early driver mutations from subsequent passenger mutations is key to molecular sub-typing of cancers, and the discovery of novel biomarkers. The availability of genomics technologies (mainly wholegenome and exome sequencing, and transcript sampling via RNA-seq, collectively referred to as NGS) have fueled recent studies on somatic mutation discovery. However, the vision is challenged by the complexity, redundancy, and errors in genomic data, and the difficulty of investigating the proteome using only genomic approaches. Recently, combination of proteomic and genomic technologies are increasingly employed. However, the complexity and redundancy of NGS data remains a challenge for proteogenomics, and various trade-offs must be made to allow for the searches to take place. This paperprovides a discussion of two such trade-offs, relating to large database search, and FDR calculations, and their implication to cancer proteogenomics. Moreover, it extends and develops the idea of a unified genomic variant database that can be searched by any mass spectrometry sample. A total of 879 BAM files downloaded from TCGA repository were used to create a 4.34 GB unified FASTA database which contained 2,787,062 novel splice junctions, 38,464 deletions, 1105 insertions, and 182,302 substitutions. Proteomic data from a single ovarian carcinoma sample (439,858 spectra) was searched against the database. By applying the most conservative FDR measure, we have identified 524 novel peptides and 65,578 known peptides at 1% FDR threshold. The novel peptides include interesting examples of doubly mutated peptides, frame-shifts, and non-sample-recruited mutations, which emphasize the strength of our approach.

  14. Identification of a Peptide from Mammal Albumins Responsible for Enhanced Pigment Production by Group B Streptococci

    PubMed Central

    Rosa-Fraile, Manuel; Sampedro, Antonio; Varela, Javier; Garcia-Peña, Marisa; Gimenez-Gallego, Guillermo

    1999-01-01

    The peptide from peptones responsible for enhanced pigment production by Streptococcus agalactiae in culture media has been isolated from a peptic digest of human albumin and has been identified as Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe. The related heptapeptide lacking the N-terminal Ile also had pigment-enhancing activity. A sequence similarity search showed that these sequences are present only in mammal albumins. PMID:10225848

  15. Identification of the structural determinants for anticancer activity of a ruthenium arene peptide conjugate.

    PubMed

    Meier, Samuel M; Novak, Maria; Kandioller, Wolfgang; Jakupec, Michael A; Arion, Vladimir B; Metzler-Nolte, Nils; Keppler, Bernhard K; Hartinger, Christian G

    2013-07-01

    Organometallic Ru(arene)-peptide bioconjugates with potent in vitro anticancer activity are rare. We have prepared a conjugate of a Ru(arene) complex with the neuropeptide [Leu(5)]-enkephalin. [Chlorido(η(6)-p-cymene)(5-oxo-κO-2-{(4-[(N-tyrosinyl-glycinyl-glycinyl-phenylalanyl-leucinyl-NH2)propanamido]-1H-1,2,3-triazol-1-yl)methyl}-4H-pyronato-κO)ruthenium(II)] (8) shows antiproliferative activity in human ovarian carcinoma cells with an IC50 value as low as 13 μM, whereas the peptide or the Ru moiety alone are hardly cytotoxic. The conjugation strategy for linking the Ru(cym) (cym=η(6)-p-cymene) moiety to the peptide involved N-terminal modification of an alkyne-[Leu(5)]-enkephalin with a 2-(azidomethyl)-5-hydroxy-4H-pyran-4-one linker, using Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC), and subsequent metallation with the Ru(cym) moiety. The ruthenium-bioconjugate was characterized by high resolution top-down electrospray ionization mass spectrometry (ESI-MS) with regard to peptide sequence, linker modification and metallation site. Notably, complete sequence coverage was obtained and the Ru(cym) moiety was confirmed to be coordinated to the pyronato linker. The ruthenium-bioconjugate was analyzed with respect to cytotoxicity-determining constituents, and through the bioconjugate models [{2-(azidomethyl)-5-oxo-κO-4H-pyronato-κO}chloride (η(6)-p-cymene)ruthenium(II)] (5) and [chlorido(η(6)-p-cymene){5-oxo-κO-2-([(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl]methyl)-4H-pyronato-κO}ruthenium(II)] (6) the Ru(cym) fragment with a triazole-carrying pyronato ligand was identified as the minimal unit required to achieve in vitro anticancer activity.

  16. Identification of a genetic locus responsible for antimicrobial peptide resistance in Clostridium difficile.

    PubMed

    McBride, Shonna M; Sonenshein, Abraham L

    2011-01-01

    Clostridium difficile causes chronic intestinal disease, yet little is understood about how the bacterium interacts with and survives in the host. To colonize the intestine and cause persistent disease, the bacterium must circumvent killing by host innate immune factors, such as cationic antimicrobial peptides (CAMPs). In this study, we investigated the effect of model CAMPs on growth and found that C. difficile is not only sensitive to these compounds but also responds to low levels of CAMPs by expressing genes that lead to CAMP resistance. By plating the bacterium on medium containing the CAMP nisin, we isolated a mutant capable of growing in three times the inhibitory concentration of CAMPs. This mutant also showed increased resistance to the CAMPs gallidermin and polymyxin B, demonstrating tolerance to different types of antimicrobial peptides. We identified the mutated gene responsible for the resistance phenotype as CD1352. This gene encodes a putative orphan histidine kinase that lies adjacent to a predicted ABC transporter operon (CD1349 to CD1351). Transcriptional analysis of the ABC transporter genes revealed that this operon was upregulated in the presence of nisin in wild-type cells and was more highly expressed in the CD1352 mutant. The insertional disruption of the CD1349 gene resulted in significant decreases in resistance to the CAMPs nisin and gallidermin but not polymyxin B. Because of their role in cationic antimicrobial peptide resistance, we propose the designation cprABC for genes CD1349 to CD1351 and cprK for the CD1352 gene. These results provide the first evidence of a C. difficile gene associated with antimicrobial peptide resistance. PMID:20974818

  17. Proteogenomic strategies for identification of aberrant cancer peptides using large-scale Next Generation Sequencing data

    DOE PAGES

    Woo, Sunghee; Cha, Seong Won; Na, Seungjin; Guest, Clark; Liu, Tao; Smith, Richard D.; Rodland, Karin D.; Payne, Samuel H.; Bafna, Vineet

    2014-11-17

    Cancer is driven by the acquisition of somatic DNA lesions. Distinguishing the early driver mutations from subsequent passenger mutations is key to molecular sub-typing of cancers, and the discovery of novel biomarkers. The availability of genomics technologies (mainly wholegenome and exome sequencing, and transcript sampling via RNA-seq, collectively referred to as NGS) have fueled recent studies on somatic mutation discovery. However, the vision is challenged by the complexity, redundancy, and errors in genomic data, and the difficulty of investigating the proteome using only genomic approaches. Recently, combination of proteomic and genomic technologies are increasingly employed. However, the complexity and redundancymore » of NGS data remains a challenge for proteogenomics, and various trade-offs must be made to allow for the searches to take place. This paperprovides a discussion of two such trade-offs, relating to large database search, and FDR calculations, and their implication to cancer proteogenomics. Moreover, it extends and develops the idea of a unified genomic variant database that can be searched by any mass spectrometry sample. A total of 879 BAM files downloaded from TCGA repository were used to create a 4.34 GB unified FASTA database which contained 2,787,062 novel splice junctions, 38,464 deletions, 1105 insertions, and 182,302 substitutions. Proteomic data from a single ovarian carcinoma sample (439,858 spectra) was searched against the database. By applying the most conservative FDR measure, we have identified 524 novel peptides and 65,578 known peptides at 1% FDR threshold. The novel peptides include interesting examples of doubly mutated peptides, frame-shifts, and non-sample-recruited mutations, which emphasize the strength of our approach.« less

  18. Spectroscopic Identification of Cyclic Imide b2-Ions from Peptides Containing Gln and Asn Residues

    NASA Astrophysics Data System (ADS)

    Grzetic, Josipa; Oomens, Jos

    2013-08-01

    In mass-spectrometry based peptide sequencing, formation of b- and y-type fragments by cleavage of the amide C-N bond constitutes the main dissociation pathway of protonated peptides under low-energy collision induced dissociation (CID). The structure of the b 2 fragment ion from peptides containing glutamine (Gln) and asparagine (Asn) residues is investigated here by infrared ion spectroscopy using the free electron laser FELIX. The spectra are compared with theoretical spectra calculated using density functional theory for different possible isomeric structures as well as to experimental spectra of synthesized model systems. The spectra unambiguously show that the b2-ions do not possess the common oxazolone structure, nor do they possess the alternative diketopiperazine structure. Instead, cyclic imide structures are formed through nucleophilic attack by the amide nitrogen atom of the Gln and Asn side chains. The alternative pathway involving nucleophilic attack from the side-chain amide oxygen atom leading to cyclic isoimide structures, which had been suggested by several authors, can clearly be excluded based on the present IR spectra. This mechanism is perhaps surprising as the amide oxygen atom is considered to be the better nucleophile; however, computations show that the products formed via attack by the amide nitrogen are considerably lower in energy. Hence, b2-ions with Asn or Gln in the second position form structures with a five-membered succinimide or a six-membered glutarimide ring, respectively. b2-Ions formed from peptides with Asn in the first position are spectroscopically shown to possess the classical oxazolone structure.

  19. Molecular identification of poisonous mushrooms using nuclear ITS region and peptide toxins: a retrospective study on fatal cases in Thailand.

    PubMed

    Parnmen, Sittiporn; Sikaphan, Sujitra; Leudang, Siriwan; Boonpratuang, Thitiya; Rangsiruji, Achariya; Naksuwankul, Khwanruan

    2016-02-01

    Cases of mushroom poisoning in Thailand have increased annually. During 2008 to 2014, the cases reported to the National Institute of Health included 57 deaths; at least 15 died after ingestion of amanitas, the most common lethal wild mushrooms inhabited. Hence, the aims of this study were to identify mushroom samples from nine clinically reported cases during the 7-year study period based on nuclear ITS sequence data and diagnose lethal peptide toxins using a reversed phase LC-MS method. Nucleotide similarity was identified using BLAST search of the NCBI database and the Barcode of Life Database (BOLD). Clade characterization was performed by maximum likelihood and Bayesian phylogenetic approaches. Based on BLAST and BOLD reference databases our results yielded high nucleotide similarities of poisonous mushroom samples to A. exitialis and A. fuliginea. Detailed phylogenetic analyses showed that all mushroom samples fall into their current classification. Detection of the peptide toxins revealed the presence of amatoxins and phallotoxins in A. exitialis and A. fuliginea. In addition, toxic α-amanitin was identified in a new provisional species, Amanita sp.1, with the highest toxin quantity. Molecular identification confirmed that the mushrooms ingested by the patients were members of the lethal amanitas in the sections Amanita and Phalloideae. In Thailand, the presence of A. exitialis was reported here for the first time and all three poisonous mushroom species provided new and informative data for clinical studies.

  20. Isolation and identification of antioxidant peptides from enzymatically hydrolyzed rice bran protein.

    PubMed

    Wattanasiritham, Ladda; Theerakulkait, Chockchai; Wickramasekara, Samanthi; Maier, Claudia S; Stevens, Jan F

    2016-02-01

    Khao Dawk Mali 105 rice bran protein (RBP) was fractionated into albumin (12.5%), globulin (13.9%), glutelin (70.8%) and prolamine (2.9%). The native and denatured RBP fractions were hydrolyzed with papain and trypsin for 3h at optimum conditions. The RBP fractions and their hydrolysates were evaluated for their antioxidant activity by the Oxygen Radical Absorbance Capacity (ORAC) assay. The trypsin-hydrolyzed denatured albumin exhibited the highest antioxidant activity with an ORAC value of 4.07 μmol of Trolox equivalent (TE)/mg protein. This hydrolysate was separated by using RP-HPLC and three fractions with high antioxidant activity were examined by LTQ-FTICR ESI mass spectrometry. The MW of the peptides from these fractions were 800-2100 Da. and consisted of 6-21 amino acid residues. Most of the peptides from the fractions demonstrated typical characteristics of well-known antioxidant peptides. The results suggest that trypsin-hydrolyzed denatured rice bran albumin might be useful as a natural food antioxidant. PMID:26304333

  1. Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition*

    PubMed Central

    Civra, Andrea; Giuffrida, Maria Gabriella; Donalisio, Manuela; Napolitano, Lorenzo; Takada, Yoshikazu; Coulson, Barbara S.; Conti, Amedeo; Lembo, David

    2015-01-01

    Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2β1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2β1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding. PMID:25814665

  2. Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics.

    PubMed

    Biniossek, Martin L; Niemer, Melanie; Maksimchuk, Ken; Mayer, Bettina; Fuchs, Julian; Huesgen, Pitter F; McCafferty, Dewey G; Turk, Boris; Fritz, Guenther; Mayer, Jens; Haecker, Georg; Mach, Lukas; Schilling, Oliver

    2016-07-01

    We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2'. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3'. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories. PMID:27122596

  3. Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics.

    PubMed

    Biniossek, Martin L; Niemer, Melanie; Maksimchuk, Ken; Mayer, Bettina; Fuchs, Julian; Huesgen, Pitter F; McCafferty, Dewey G; Turk, Boris; Fritz, Guenther; Mayer, Jens; Haecker, Georg; Mach, Lukas; Schilling, Oliver

    2016-07-01

    We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2'. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3'. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories.

  4. Identification of a Peptide-Pheromone that Enhances Listeria monocytogenes Escape from Host Cell Vacuoles

    PubMed Central

    Xayarath, Bobbi; Alonzo, Francis; Freitag, Nancy E.

    2015-01-01

    Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol. PMID:25822753

  5. Identification and molecular characterization of oat peptides implicated on coeliac immune response

    PubMed Central

    Comino, Isabel; Bernardo, David; Bancel, Emmanuelle; Moreno, María de Lourdes; Sánchez, Borja; Barro, Francisco; Šuligoj, Tanja; Ciclitira, Paul J.; Cebolla, Ángel; Knight, Stella C.; Branlard, Gérard; Sousa, Carolina

    2016-01-01

    Background Oats provide important nutritional and pharmacological properties, although their safety in coeliac patients remains controversial. Previous studies have confirmed that the reactivity of the anti-33-mer monoclonal antibody with different oat varieties is proportional to the immune responses in terms of T-cell proliferation. Although the impact of these varieties on the adaptive response has been studied, the role of the dendritic cells (DC) is still poorly understood. The aim of this study is to characterize different oat fractions and to study their effect on DC from coeliac patients. Methods and results Protein fractions were isolated from oat grains and analyzed by SDS–PAGE. Several proteins were characterized in the prolamin fraction using immunological and proteomic tools, and by Nano-LC-MS/MS. These proteins, analogous to α- and γ-gliadin-like, showed reactive sequences to anti-33-mer antibody suggesting their immunogenic potential. That was further confirmed as some of the newly identified oat peptides had a differential stimulatory capacity on circulating DC from coeliac patients compared with healthy controls. Conclusions This is the first time, to our knowledge, where newly identified oat peptides have been shown to elicit a differential stimulatory capacity on circulating DC obtained from coeliac patients, potentially identifying immunogenic properties of these oat peptides. PMID:26853779

  6. Identification of a peptide-pheromone that enhances Listeria monocytogenes escape from host cell vacuoles.

    PubMed

    Xayarath, Bobbi; Alonzo, Francis; Freitag, Nancy E

    2015-03-01

    Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol.

  7. Identification of a Novel Proline-Rich Antimicrobial Peptide from Brassica napus.

    PubMed

    Cao, Huihui; Ke, Tao; Liu, Renhu; Yu, Jingyin; Dong, Caihua; Cheng, Mingxing; Huang, Junyan; Liu, Shengyi

    2015-01-01

    Proline-rich antimicrobial peptides (PR-AMPs) are a group of cationic host defense peptides that are characterized by a high content of proline residues. Up to now, they have been reported in some insects, vertebrate and invertebrate animals, but are not found in plants. In this study, we performed an in silico screening of antimicrobial peptides, which led to discovery of a Brassica napus gene encoding a novel PR-AMP. This gene encodes a 35-amino acid peptide with 13 proline residues, designated BnPRP1. BnPRP1 has 40.5% identity with a known proline-rich antimicrobial peptide SP-B from the pig. BnPRP1 was artificially synthetized and cloned into the prokaryotic expression vector pET30a/His-EDDIE-GFP. Recombinant BnPRP1 was produced in Escherichia coli and has a predicted molecular mass of 3.8 kDa. Analysis of its activity demonstrated that BnPRP1 exhibited strong antimicrobial activity against Gram-positive bacterium, Gram-negative bacterium, yeast and also had strong antifungal activity against several pathogenic fungi, such as Sclerotinia sclerotiorum, Mucor sp., Magnaporthe oryzae and Botrytis cinerea. Circular dichroism (CD) revealed the main secondary structure of BnPRP1 was the random coil. BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation. At 48 hours after S. sclerotiorum inoculation, the expression of BnPRP1 increased significantly in the susceptible lines while slight decrease occurred in resistant lines. These suggested that BnPRP1 might play a role in the plant defense response against S. sclerotiorum. BnPRP1 isolated from B. napus was the first PR-AMP member that was characterized in plants, and its homology sequences were found in some other Brassicaceae plants by the genome sequences analysis. Compared with the known PR-AMPs, BnPRP1 has the different primary sequences and antimicrobial activity. Above all, this study gives a chance to cast a new light on further understanding about the AMPs' mechanism and application.

  8. Identification of a Novel Proline-Rich Antimicrobial Peptide from Brassica napus.

    PubMed

    Cao, Huihui; Ke, Tao; Liu, Renhu; Yu, Jingyin; Dong, Caihua; Cheng, Mingxing; Huang, Junyan; Liu, Shengyi

    2015-01-01

    Proline-rich antimicrobial peptides (PR-AMPs) are a group of cationic host defense peptides that are characterized by a high content of proline residues. Up to now, they have been reported in some insects, vertebrate and invertebrate animals, but are not found in plants. In this study, we performed an in silico screening of antimicrobial peptides, which led to discovery of a Brassica napus gene encoding a novel PR-AMP. This gene encodes a 35-amino acid peptide with 13 proline residues, designated BnPRP1. BnPRP1 has 40.5% identity with a known proline-rich antimicrobial peptide SP-B from the pig. BnPRP1 was artificially synthetized and cloned into the prokaryotic expression vector pET30a/His-EDDIE-GFP. Recombinant BnPRP1 was produced in Escherichia coli and has a predicted molecular mass of 3.8 kDa. Analysis of its activity demonstrated that BnPRP1 exhibited strong antimicrobial activity against Gram-positive bacterium, Gram-negative bacterium, yeast and also had strong antifungal activity against several pathogenic fungi, such as Sclerotinia sclerotiorum, Mucor sp., Magnaporthe oryzae and Botrytis cinerea. Circular dichroism (CD) revealed the main secondary structure of BnPRP1 was the random coil. BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation. At 48 hours after S. sclerotiorum inoculation, the expression of BnPRP1 increased significantly in the susceptible lines while slight decrease occurred in resistant lines. These suggested that BnPRP1 might play a role in the plant defense response against S. sclerotiorum. BnPRP1 isolated from B. napus was the first PR-AMP member that was characterized in plants, and its homology sequences were found in some other Brassicaceae plants by the genome sequences analysis. Compared with the known PR-AMPs, BnPRP1 has the different primary sequences and antimicrobial activity. Above all, this study gives a chance to cast a new light on further understanding about the AMPs' mechanism and application

  9. Identification of novel dipeptidyl peptidase-IV and angiotensin-I-converting enzyme inhibitory peptides from meat proteins using in silico analysis.

    PubMed

    Lafarga, Tomas; O'Connor, Paula; Hayes, Maria

    2014-09-01

    Angiotensin-I-converting enzyme (ACE-I, EC 3.4.15.1), renin (EC 3.4.23.15), and dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5) play key roles in the control of hypertension and the development of type-2 diabetes and other diseases associated with metabolic syndrome. The aim of this work was to utilize known in silico methodologies, peptide databases and software including ProtParam (http://web.expasy.org/protparam/), Basic Local Alignment Tool (BLAST), ExPASy PeptideCutter (http://web.expasy.org/peptide_cutter/) and BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep) to assess the release of potentially bioactive DPP-IV, renin and ACE-I inhibitory peptides from bovine and porcine meat proteins including hemoglobin, collagen and serum albumin. These proteins were chosen as they are found commonly in meat by-products such as bone, blood and low-value meat cuts. In addition, the bioactivities of identified peptides were confirmed using chemical synthesis and in vitro bioassays. The concentration of peptide required to inhibit the activity of ACE-I and DPP-IV by 50% was determined for selected, active peptides. Novel ACE-I and DPP-IV inhibitory peptides were identified in this study using both in silico analysis and a literature search to streamline enzyme selection for peptide production. These novel peptides included the ACE-I inhibitory tri-peptide Ile-Ile-Tyr and the DPP-IV inhibitory tri-peptide Pro-Pro-Leu corresponding to sequences f (182-184) and f (326-328) of both porcine and bovine serum albumin which can be released following hydrolysis with the enzymes papain and pepsin, respectively. This work demonstrates that meat proteins are a suitable resource for the generation of bioactive peptides and further demonstrates the usefulness of in silico methodologies to streamline identification and generation of bioactive peptides. PMID:25020248

  10. "Fahrenheit 9-11," Need for Closure and the Priming of Affective Ambivalence: An Assessment of Intra-Affective Structures by Party Identification

    ERIC Educational Resources Information Center

    Holbert, R. Lance; Hansen, Glenn J.

    2006-01-01

    This study extends priming research in political communication by focusing on an alternative political information source (i.e., Michael Moore's Fahrenheit 9-11), affect rather than cognitions, and the existence of intra-affective ambivalence. In addition, two moderator variables are analyzed: political party identification and need for closure.…

  11. Anti-HIV screening for cell-penetrating peptides using chloroquine and identification of anti-HIV peptides derived from matrix proteins.

    PubMed

    Mizuguchi, Takaaki; Ohashi, Nami; Nomura, Wataru; Komoriya, Mao; Hashimoto, Chie; Yamamoto, Naoki; Murakami, Tsutomu; Tamamura, Hirokazu

    2015-08-01

    Previously, compounds which inhibit the HIV-1 replication cycle were found in overlapping peptide libraries covering the whole sequence of an HIV-1 matrix (MA) protein constructed with the addition of an octa-arginyl group. The two top lead compounds are sequential fragments MA-8L and MA-9L. In the present study, the addition of chloroquine in cell-based anti-HIV assays was proven to be an efficient method with which to find anti-HIV compounds among several peptides conjugated by cell-penetrating signals such as an octa-arginyl group: the conjugation of an octa-arginyl group to individual peptides contained in whole proteins in combination with the addition of chloroquine in cells is a useful assay method to search active peptides. To find more potent fragment peptides, individual peptides between MA-8L and MA-9L, having the same peptide chain length but with sequences shifted by one amino acid residue, were synthesized in this paper and their anti-HIV activity was evaluated with an anti-HIV assay using chloroquine. As a result, the peptides in the C-terminal side of the series, which are relatively close to MA-9L, showed more potent inhibitory activity against both X4-HIV-1 and R5-HIV-1 than the peptides in the N-terminal side.

  12. Identification of a Peptide Toxin from Grammostola spatulata Spider Venom That Blocks Cation-Selective Stretch-Activated Channels

    PubMed Central

    Suchyna, Thomas M.; Johnson, Janice H.; Hamer, Katherine; Leykam, Joseph F.; Gage, Douglas A.; Clemo, Henry F.; Baumgarten, Clive M.; Sachs, Frederick

    2000-01-01

    We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance (∼45 pS at −100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of ∼630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces ∼40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation. PMID:10779316

  13. Identification of peptidic inhibitors of the alternative complement pathway based on Staphylococcus aureus SCIN proteins.

    PubMed

    Summers, Brady J; Garcia, Brandon L; Woehl, Jordan L; Ramyar, Kasra X; Yao, Xiaolan; Geisbrecht, Brian V

    2015-10-01

    The complement system plays a central role in a number of human inflammatory diseases, and there is a significant need for development of complement-directed therapies. The discovery of an arsenal of anti-complement proteins secreted by the pathogen Staphylococcus aureus brought with it the potential for harnessing the powerful inhibitory properties of these molecules. One such family of inhibitors, the SCINs, interact with a functional "hot-spot" on the surface of C3b. SCINs not only stabilize an inactive form of the alternative pathway (AP) C3 convertase (C3bBb), but also overlap the C3b binding site of complement factors B and H. Here we determined that a conserved Arg residue in SCINs is critical for function of full-length SCIN proteins. Despite this, we also found SCIN-specific differences in the contributions of other residues found at the C3b contact site, which suggested that a more diverse repertoire of residues might be able to recognize this region of C3b. To investigate this possibility, we conducted a phage display screen aimed at identifying SCIN-competitive 12-mer peptides. In total, seven unique sequences were identified and all exhibited direct C3b binding. A subset of these specifically inhibited the AP in assays of complement function. The mechanism of AP inhibition by these peptides was probed through surface plasmon resonance approaches, which revealed that six of the seven peptides disrupted C3bBb formation by interfering with factor B/C3b binding. To our knowledge this study has identified the first small molecules that retain inhibitory properties of larger staphylococcal immune evasion proteins.

  14. Identification of novel peptide substrates for protein farnesyltransferase reveals two substrate classes with distinct sequence selectivities

    PubMed Central

    Hougland, James L.; Hicks, Katherine A.; Hartman, Heather L.; Kelly, Rebekah A.; Watt, Terry J.; Fierke, Carol A.

    2010-01-01

    Prenylation is a post-translational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development and farnesyltransferase inhibitors (FTIs) are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for FTI efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover conditions. Based on these results, a second library was designed that yielded an additional 29 novel multiple-turnover FTase substrates and 45 single-turnover substrates. The two classes of substrates exhibit different specificity requirements. Efficient multiple-turnover reactivity correlates with the presence of a nonpolar amino acid at the a2 position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca1a2X sequence model. In contrast, the sequences of the single-turnover substrates vary significantly more at both the a2 and X residues and are not well-described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets

  15. Identification of IgE sequential epitopes of lentil (Len c 1) by peptide microarray immunoassay

    PubMed Central

    Vereda, Andrea; Andreae, Doerthe A.; Lin, Jing; Shreffler, Wayne G.; Ibañez, Maria Dolores; Cuesta-Herranz, Javier; Bardina, Luda; Sampson, Hugh A.

    2010-01-01

    Background Lentils are oftentimes responsible for allergic reactions to legumes in Mediterranean children. Though the primary sequence of the major allergen, Len c 1 is known, the location of the IgE binding epitopes remains undefined. Objective We sought to identify IgE-binding epitopes of Len c 1 and relate epitope binding to clinical characteristics. Methods 135 peptides corresponding to the primary sequence of Len c 1 were probed with sera from 33 lentil-allergic individuals and 15 non-atopic controls by means of microarray immunoassay. Lentil-specific IgE, Skin Prick Tests and clinical reactions to lentil were determined. Epitopes were defined as overlapping signal above inter- and intra-slide cut-offs and confirmed by inhibition assays using a peptide from the respective region. Hierarchical clustering of microarray data was used to correlate binding patterns with clinical findings. Results The lentil-allergic patients specifically recognized IgE-binding epitopes located in the C-terminal region, between peptide 107 and 135. Inhibition experiments confirmed the specificity of IgE binding in this region, identifying different epitopes. Linkage of cluster results with clinical data and lentil specific IgE levels displayed a positive correlation between lentil-specific IgE levels, epitope recognition and respiratory symptoms. Modeling based on the three-dimensional structure of a homologous soy vicilin suggests that the Len c 1 epitopes identified are exposed on the surface of the molecule. Conclusion Several IgE-binding sequential epitopes of Len c 1 have been identified. Epitopes are located in the C-terminal region, and are predicted to be exposed on the surface of the protein. Epitope diversity is positively correlated with IgE levels, pointing to a more polyclonal IgE response. PMID:20816193

  16. Peptide nucleic acid fluorescence in situ hybridization for identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii.

    PubMed

    Zhang, Xiaofeng; Wu, Shan; Li, Ke; Shuai, Jiangbing; Dong, Qiang; Fang, Weihuan

    2012-07-01

    A fluorescent in situ hybridization (FISH) method in conjunction with fluorescin-labeled peptide nucleic acid (PNA) probes (PNA-FISH) for detection of Listeria species was developed. In silico analysis showed that three PNA probes Lis-16S-1, Lm-16S-2 and Liv-16S-5 were suitable for specific identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii, respectively. These probes were experimentally verified by their reactivity against 19 strains of six Listeria species (excluding newly described species Listeria marthii and Listeria rocourtiae) and eight other bacterial species. The PNA-FISH method was optimized as 30 min of hybridization with 0.2% Triton X-100 in the solution and used to identify 85 Listeria strains from individual putative Listeria colonies on PALCAM agar plates streaked from selectively enriched cultures of 780 food or food-related samples. Of the 85 Listeria strains, thirty-seven were identified as L. monocytogenes with the probe Lm-16S-2 and two as L. ivanovii with the probe Liv-16S-5 which was in agreement with the results obtained by the API LISTERIA method. Thus, the PNA-FISH protocol has the potential for identification of pathogenic Listeria spp. from food or food-related samples.

  17. Supporting data for the MS identification of distinct transferrin glycopeptide glycoforms and citrullinated peptides associated with inflammation or autoimmunity

    PubMed Central

    Rosal-Vela, A.; Barroso, A.; Giménez, E.; García-Rodríguez, S.; Longobardo, V.; Postigo, J.; Iglesias, M.; Lario, A.; Merino, J.; Merino, R.; Zubiaur, M.; Sanz-Nebot, V.; Sancho, J.

    2016-01-01

    This data article presents the results of all the statistical analyses applied to the relative intensities of the detected 2D-DiGE protein spots for each of the 3 performed DiGE experiments. The data reveals specific subsets of protein spots with significant differences between WT and CD38-deficient mice with either Collagen-induced arthritis (CIA), or with chronic inflammation induced by CFA, or under steady-state conditions. This article also shows the MS data analyses that allowed the identification of the protein species which serve to discriminate the different experimental groups used in this study. Moreover, the article presents MS data on the citrullinated peptides linked to specific protein species that were generated in CIA+ or CFA-treated mice. Lastly, this data article provides MS data on the efficiency of the analyses of the transferrin (Tf) glycopeptide glycosylation pattern in spleen and serum from CIA+ mice and normal controls. The data supplied in this work is related to the research article entitled “identification of multiple transferrin species in spleen and serum from mice with collagen-induced arthritis which may reflect changes in transferrin glycosylation associated with disease activity: the role of CD38” [1]. All mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifiers PRIDE: PXD002644, PRIDE: PXD002643, PRIDE: PXD003183 and PRIDE: PXD003163. PMID:26909372

  18. Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH).

    PubMed

    Malic, Sladjana; Hill, Katja E; Hayes, Anthony; Percival, Steven L; Thomas, David W; Williams, David W

    2009-08-01

    Biofilms provide a reservoir of potentially infectious micro-organisms that are resistant to antimicrobial agents, and their importance in the failure of medical devices and chronic inflammatory conditions is increasingly being recognized. Particular research interest exists in the association of biofilms with wound infection and non-healing, i.e. chronic wounds. In this study, fluorescent in situ hybridization (FISH) was used in combination with confocal laser scanning microscopy (CLSM) to detect and characterize the spatial distribution of biofilm-forming bacteria which predominate within human chronic skin wounds (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sp. and Micrococcus sp.). In vitro biofilms were prepared using a constant-depth film fermenter and a reconstituted human epidermis model. In vivo biofilms were also studied using biopsy samples from non-infected chronic venous leg ulcers. The specificity of peptide nucleic acid (PNA) probes for the target organisms was confirmed using mixed preparations of planktonic bacteria and multiplex PNA probing. Identification and location of individual bacterial species within multi-species biofilms demonstrated that P. aeruginosa was predominant. CLSM revealed clustering of individual species within mixed-species biofilms. FISH analysis of archive chronic wound biopsy sections showed bacterial presence and allowed bacterial load to be determined. The application of this standardized procedure makes available an assay for identification of single- or multi-species bacterial populations in tissue biopsies. The technique provides a reliable tool to study bacterial biofilm formation and offers an approach to assess targeted biofilm disruption strategies in vivo. PMID:19477903

  19. Detection and identification of specific bacteria in wound biofilms using peptide nucleic acid fluorescent in situ hybridization (PNA FISH).

    PubMed

    Malic, Sladjana; Hill, Katja E; Hayes, Anthony; Percival, Steven L; Thomas, David W; Williams, David W

    2009-08-01

    Biofilms provide a reservoir of potentially infectious micro-organisms that are resistant to antimicrobial agents, and their importance in the failure of medical devices and chronic inflammatory conditions is increasingly being recognized. Particular research interest exists in the association of biofilms with wound infection and non-healing, i.e. chronic wounds. In this study, fluorescent in situ hybridization (FISH) was used in combination with confocal laser scanning microscopy (CLSM) to detect and characterize the spatial distribution of biofilm-forming bacteria which predominate within human chronic skin wounds (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sp. and Micrococcus sp.). In vitro biofilms were prepared using a constant-depth film fermenter and a reconstituted human epidermis model. In vivo biofilms were also studied using biopsy samples from non-infected chronic venous leg ulcers. The specificity of peptide nucleic acid (PNA) probes for the target organisms was confirmed using mixed preparations of planktonic bacteria and multiplex PNA probing. Identification and location of individual bacterial species within multi-species biofilms demonstrated that P. aeruginosa was predominant. CLSM revealed clustering of individual species within mixed-species biofilms. FISH analysis of archive chronic wound biopsy sections showed bacterial presence and allowed bacterial load to be determined. The application of this standardized procedure makes available an assay for identification of single- or multi-species bacterial populations in tissue biopsies. The technique provides a reliable tool to study bacterial biofilm formation and offers an approach to assess targeted biofilm disruption strategies in vivo.

  20. Identification of T- and B-cell epitopes of the S2 and S3 subunits of pertussis toxin by use of synthetic peptides.

    PubMed Central

    Chong, P; Zobrist, G; Sia, C; Loosmore, S; Klein, M

    1992-01-01

    To design an optimized synthetic vaccine against whooping cough, we have studied the biological and immunological properties of three peptides of the S2 subunit and nine overlapping synthetic peptides covering the entire sequence of the S3 subunit of pertussis toxin (PT). Synthetic peptides corresponding to sequences 18 to 41, 78 to 108, 134 to 154, and 149 to 176 of S3 were found to be consistently capable of stimulating the proliferation of PT-specific T-cell lines primed with pertussis toxoid in both BALB/c and A/J strains of mice. All synthetic peptides were recognized by rabbit antisera raised against PT or pertussis toxoid. Both S2 and S3 peptide-keyhole limpet hemocyanin (KLH) conjugates in the presence of complete Freund's adjuvant induced peptide-specific antibody responses in rabbits, and the antisera raised against S2(1-23), S3(18-41), S3(37-64), and S3(149-176) peptide-KLH conjugates cross-reacted with both subunits in the immunoblots. All antisera except those against S2(123-154) and S3(103-127) reacted with native PT in an enzyme-linked immunosorbent assay (ELISA) with PT directly coated onto microtiter wells. In contrast, antisera raised against S2(123-154), S3(1-23), S3(18-41), S3(37-64), S3(60-87), and S3(103-127) peptide-KLH conjugates recognized native PT in a fetuin-PT capture ELISA. S2(78-98), S3(1-23), and S3(149-176) peptide-KLH conjugates elicited good PT-neutralizing antibody responses as judged by the antitoxin CHO cell assay. Identification of these B-cell neutralization epitopes and T-cell immunodominant determinants represents a first step towards the rational design of a synthetic vaccine against whooping cough. PMID:1383153

  1. Identification of a novel antimicrobial peptide from Brazilian coast coral Phyllogorgia dilatata.

    PubMed

    de Lima, Loiane Alves; Migliolo, Ludovico; Barreiro e Castro, Clovis; Pires, Debora de Oliveira; Lopez-Abarrategui, Carlos; Gonçalves, Eveline Ferreira; Vasconcelos, Ilka Maria; de Oliveira, Jose Tadeu Abreu; Otero-Gonzalez, Anselmo de Jesus; Franco, Octávio Luiz; Dias, Simoni Campos

    2013-10-01

    The marine ecosystem is able to provide enormous biomolecule diversity that could be used for treatment of various diseases. In this highly competitive environment, organisms need chemical barriers to reduce or avoid microorganism contamination. Among the molecules that protect these animals the antimicrobial peptides (AMPs) are included. In the present study, crude extracts of coral coral specimens Carijoa riisei, Muriceopsis sulphurea, Neospongodes atlantica, Palythoa caribeorum, Phyllogorgia dilatata and Plexaurella grandiflora were challenged against multiple Grampositive and -negative bacteria showing different activities. P. dilatata crude extract showed the antibacterial activity, and was ammonium-sulfate (0-40%) fractionated, being able to control the growth of K. pneumoniae, S. flexineri and S. aureus. Rich-fraction was further purified by using Amicon® Ultra Centrifugal 10 kDa associated with reversed-phase HPLC chromatography (C18), producing the peptide named Pd-AMP1. Pd-AMP1 was able to inhibit S. aureus development. Mass spectrometry analyses showed a monoisotopic mass of 5372.66 Da and N-terminal sequence showed no significant match with databank. In this view, the prospecting of protein biomolecules and biotechnological potential from marine animals is still little explored and may serve as an alternative to common antibiotics.

  2. Identification of Synthetic Host Defense Peptide Mimics That Exert Dual Antimicrobial and Anti-Inflammatory Activities

    PubMed Central

    Som, Abhigyan; Navasa, Nicolás; Percher, Avital; Scott, Richard W.

    2012-01-01

    A group of synthetic antimicrobial oligomers, inspired by naturally occurring antimicrobial peptides, were analyzed for the ability to modulate innate immune responses to Toll-like receptor (TLR) ligands. These synthetic mimics of antimicrobial peptides (SMAMPs) specifically reduced cytokine production in response to Staphylococcus aureus and the S. aureus component lipoteichoic acid (LTA), a TLR2 agonist. Anti-inflammatory SMAMPs prevented the induction of tumor necrosis factor (TNF), interleukin 6 (IL-6), and IL-10 in response to S. aureus or LTA, but no other TLR2 ligands. We show that these SMAMPs bind specifically to LTA in vitro and prevent its interaction with TLR2. Importantly, the SMAMP greatly reduced the induction of TNF and IL-6 in vivo in mice acutely infected with S. aureus while simultaneously reducing bacterial loads dramatically (4 log10). Thus, these SMAMPs can eliminate the damage induced by pathogen-associated molecular patterns (PAMPs) while simultaneously eliminating infection in vivo. They are the first known SMAMPs to demonstrate anti-inflammatory and antibacterial activities in vivo. PMID:22956655

  3. Ultrasensitive Identification of Localization Variants of Modified Peptides Using Ion Mobility Spectrometry

    SciTech Connect

    Ibrahim, Yehia M.; Shvartsburg, Alexandre A.; Smith, Richard D.; Belov, Mikhail E.

    2011-05-28

    Localization of the modification sites on peptides is challenging, particularly when multiple modifications or mixtures of localization isomers (variants) are involved. Such variants commonly coelute in liquid chromatography and may be undistinguishable in tandem mass spectrometry (MS/MS) for lack of unique fragments. Here, we have resolved the variants of singly and doubly phosphorylated peptides employing drift tube ion mobility spectrometry (IMS) coupled to time-of-flight mass spectrometry. Even with a moderate IMS resolving power of ~80, substantial separation was achieved for both 2+ and 3+ ions normally generated by electrospray ionization, including for the variant indistinguishable by MS/MS. Variants often exhibit a distribution of 3-D conformers, which can be adjusted for optimum IMS separation by prior field heating of ions in a funnel trap. The peak assignments were confirmed using MS/MS after IMS separation, but known species could be identified using just the ion mobility "tag". Avoiding the MS/MS step lowers the detection limit of localization variants to <100 attomoles, an order of magnitude better than provided by electron transfer dissociation in an Orbitrap MS.

  4. Identification and Characterization of Novel Antioxidant Peptides Involved in Redox Homeostasis of Frog, Limnonectes fragilis.

    PubMed

    Yu, Haining; Qiao, Xue; Gao, Jiuxiang; Wang, Chen; Cai, Shasha; Feng, Lan; Wang, Hui; Wang, Yi-Peng

    2015-01-01

    Previous studies have proved that a novel antioxidant system composed of various antioxidant peptides (AOPs) exists in the skin of ranid frogs, keeping the redox homeostasis. However, only a small number of AOPs have been identified so far. Here, a total of 47 cDNA sequences encoding 21 different AOPs belonging to 11 families were cloned from the skin cDNA library of Limnonectes fragilis. Among them, fragilin-A1 (VKRRGQDCIHGFCSD) and fragilin-B1 (GQFNDKRWIPFG) were also purified from skin secretions. They were selected with odorranain-Q-Lf (APIRMWYMYRKLTDMEPKPVA), the newest sequence among all 21 AOPs, to evaluate the antioxidant activities by direct free radical scavenging and lipid peroxidation inhibition assay. Results demonstrated that all peptides possessed strong DPPH and ABTS(.+) scavenging activities, and effectively inhibited lipid peroxidation in linoleic acid emulsion system during a 7- day test. No cytotoxic and hemolytic activity against human erythrocytes was observed for the three AOPs. The homology modeling analysis revealed that they all adopt tertiary structures ideally suited for the key residues to come into contact with the radicals. Current results reveal the existence of antioxidant system constituted of AOPs in the skin of the L. fragilis, and furthermore provide excellent templates for the development of novel antioxidant agents. PMID:26122987

  5. Identification and characterization of antimicrobial peptide, defensin, in the taiga tick, Ixodes persulcatus.

    PubMed

    Saito, Y; Konnai, S; Yamada, S; Imamura, S; Nishikado, H; Ito, T; Onuma, M; Ohashi, K

    2009-08-01

    Ixodes persulcatus is the primary vector for human tick-borne diseases in Japan. A cDNA library was constructed from whole body homogenates of fed nymphs of I. persulcatus. From this library, one cDNA encoding defensin-like antimicrobial peptide was identified. The amino-acid sequence showed high similarity to those of the defensins of other ticks and arthropods. I. persulcatus defensin mRNA transcripts were detected at all life cycle stages of fed ticks and found to be predominantly expressed in the midguts of adult female ticks, but not in the salivary glands, a finding corroborated by Western blotting analysis. To investigate the function of I. persulcatus defensin, we examined its antibacterial activity by evaluation of growth of several bacterial strains in the presence of the synthetic peptide. The defensin from I. persulcatus markedly inhibited the growth of Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis and Corynebacterium renale, but not Gram-negative bacteria except Escherichia coli O157. In conclusion, these results suggest that I. persulcatus defensin may be playing a significant role in the defence against microbes from bloodmeals.

  6. Identification of novel serum peptides biomarkers for female breast cancer patients in Western China.

    PubMed

    Yang, Juan; Xiong, Xiaofan; Liu, Siyuan; Zhu, Jiang; Luo, Mai; Liu, Liying; Zhao, Lingyu; Qin, Yannan; Song, Tusheng; Huang, Chen

    2016-03-01

    This study aimed to identify novel serum peptides biomarkers for female breast cancer (BC) patients. We analyzed the serum proteomic profiling of 247 serum samples from 96 BC patients, 48 additional paired pre- and postoperative BC patients, 39 fibroadenoma patients as benign disease controls, and 64 healthy controls, using magnetic-bead-based separation followed by MALDI-TOF MS. ClinProTools software identified 78 m/z peaks that differed among all analyzed groups, ten peaks were significantly different (P < 0.0001), with Peaks 1-6 upregulated and Peaks 7-10 downregulated in BC. Moreover, three peaks of ten (Peak 1, m/z: 2660.11; Peak 2, m/z: 1061.09; Peak 10, m/z: 1041.25) showed a tendency to return to healthy control values after surgery. And these three peptide biomarkers were identified as FGA605-629, ITIH4 347-356, and APOA2 43-52. Methods used in this study could generate serum peptidome profiles of BC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy. PMID:26705257

  7. Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library.

    PubMed

    Mohrlüder, Jeannine; Stangler, Thomas; Hoffmann, Yvonne; Wiesehan, Katja; Mataruga, Anja; Willbold, Dieter

    2007-11-01

    4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca(2+) -dependent chaperone and a Ca(2+) -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K(d) = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein. PMID:17916189

  8. Identification of a novel antimicrobial peptide from amphioxus Branchiostoma japonicum by in silico and functional analyses

    PubMed Central

    Liu, Haohan; Lei, Miaomiao; Du, Xiaoyuan; Cui, Pengfei; Zhang, Shicui

    2015-01-01

    The emergence of multi-drug resistant (MDR) microbes leads to urgent demands for novel antibiotics exploration. We demonstrated a cDNA from amphioxus Branchiostoma japonicum, designated Bjamp1, encoded a protein with features typical of antimicrobial peptides (AMPs), which is not homologous to any AMPs currently discovered. It was found that Bjamp1 was expressed in distinct tissues, and its expression was remarkably up-regulated following challenge with LPS and LTA. Moreover, the synthesized putative mature AMP, mBjAMP1, underwent a coil-to-helix transition in the presence of TFE or SDS, agreeing well with the expectation that BjAMP1 was a potential AMP. Functional assays showed that mBjAMP1 inhibited the growth of all the bacteria tested, and induced membrane/cytoplasmic damage. ELISA indicated that mBjAMP1 was a pattern recognition molecule capable of identifying LPS and LTA. Importantly, mBjAMP1 disrupted the bacterial membranes by a membranolytic mechanism. Additionally, mBjAMP1 was non-cytotoxic to mammalian cells. Collectively, these data indicate that mBjAMP1 is a new AMP with a high bacterial membrane selectivity, rendering it a promising template for the design of novel peptide antibiotics against MDR microbes. It also shows for the first time that use of signal conserved sequence of AMPs is effective identifying potential AMPs across different animal classes. PMID:26680226

  9. Angiotensin I-converting enzyme inhibitory activity of gelatin hydrolysates and identification of bioactive peptides.

    PubMed

    Herregods, Griet; Van Camp, John; Morel, Nicole; Ghesquière, Bart; Gevaert, Kris; Vercruysse, Lieselot; Dierckx, Stephan; Quanten, Erwin; Smagghe, Guy

    2011-01-26

    In this project we report on the angiotensin I-converting enzyme (ACE)-inhibitory activity of a bovine gelatin hydrolysate (Bh2) that was submitted to further hydrolysis by different enzymes. The thermolysin hydrolysate (Bh2t) showed the highest in vitro ACE inhibitory activity, and interestingly a marked in vivo blood pressure-lowering effect was demonstrated in spontaneously hypertensive rats (SHR). In contrast, Bh2 showed no effect in SHR, confirming the need for the extra thermolysin hydrolysis. Hence, an angiotensin I-evoked contractile response in isolated rat aortic rings was inhibited by Bh2t, but not by Bh2, suggesting ACE inhibition as the underlying antihypertensive mechanism for Bh2t. Using mass spectrometry, seven small peptides, AG, AGP, VGP, PY, QY, DY and IY or LY or HO-PY were identified in Bh2t. As these peptides showed ACE inhibitory activity and were more prominent in Bh2t than in Bh2, the current data provide evidence that these contribute to the antihypertensive effect of Bh2t.

  10. Cell growth and proteolytic activity of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus in milk as affected by supplementation with peptide fractions.

    PubMed

    Gandhi, Akanksha; Shah, Nagendra P

    2014-12-01

    The present investigation examined the effects of supplementation of milk peptide fractions produced by enzymatic hydrolysis on the fermentation of reconstituted skim milk (RSM). Changes in pH, cell growth, proteolytic activity, and angiotensin-converting enzyme (ACE)-inhibitory activity were monitored during fermentation of RSM by pure cultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus. The study showed that supplementation with peptide fractions of different molecular weights did not significantly affect the bacterial growth in RSM. All bacteria showed an increased proteolytic activity in RSM supplemented with large peptides (>10 kDa), and L. helveticus in general exhibited the highest proteolytic activity among the bacteria studied. The ACE-inhibitory activity was observed to be the maximum in RSM supplemented with larger peptides (>10 kDa) for all bacteria. The results suggest that proteolysis by bacteria leads to increased production of ACE-inhibitory peptides compared to the supplemented peptides produced by enzymatic hydrolysis.

  11. Species identification of archaeological skin objects from Danish bogs: comparison between mass spectrometry-based peptide sequencing and microscopy-based methods.

    PubMed

    Brandt, Luise Ørsted; Schmidt, Anne Lisbeth; Mannering, Ulla; Sarret, Mathilde; Kelstrup, Christian D; Olsen, Jesper V; Cappellini, Enrico

    2014-01-01

    Denmark has an extraordinarily large and well-preserved collection of archaeological skin garments found in peat bogs, dated to approximately 920 BC - AD 775. These objects provide not only the possibility to study prehistoric skin costume and technologies, but also to investigate the animal species used for the production of skin garments. Until recently, species identification of archaeological skin was primarily performed by light and scanning electron microscopy or the analysis of ancient DNA. However, the efficacy of these methods can be limited due to the harsh, mostly acidic environment of peat bogs leading to morphological and molecular degradation within the samples. We compared species assignment results of twelve archaeological skin samples from Danish bogs using Mass Spectrometry (MS)-based peptide sequencing, against results obtained using light and scanning electron microscopy. While it was difficult to obtain reliable results using microscopy, MS enabled the identification of several species-diagnostic peptides, mostly from collagen and keratins, allowing confident species discrimination even among taxonomically close organisms, such as sheep and goat. Unlike previous MS-based methods, mostly relying on peptide fingerprinting, the shotgun sequencing approach we describe aims to identify the complete extracted ancient proteome, without preselected specific targets. As an example, we report the identification, in one of the samples, of two peptides uniquely assigned to bovine foetal haemoglobin, indicating the production of skin from a calf slaughtered within the first months of its life. We conclude that MS-based peptide sequencing is a reliable method for species identification of samples from bogs. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium with the dataset identifier PXD001029.

  12. Species Identification of Archaeological Skin Objects from Danish Bogs: Comparison between Mass Spectrometry-Based Peptide Sequencing and Microscopy-Based Methods

    PubMed Central

    Brandt, Luise Ørsted; Schmidt, Anne Lisbeth; Mannering, Ulla; Sarret, Mathilde; Kelstrup, Christian D.; Olsen, Jesper V.; Cappellini, Enrico

    2014-01-01

    Denmark has an extraordinarily large and well-preserved collection of archaeological skin garments found in peat bogs, dated to approximately 920 BC – AD 775. These objects provide not only the possibility to study prehistoric skin costume and technologies, but also to investigate the animal species used for the production of skin garments. Until recently, species identification of archaeological skin was primarily performed by light and scanning electron microscopy or the analysis of ancient DNA. However, the efficacy of these methods can be limited due to the harsh, mostly acidic environment of peat bogs leading to morphological and molecular degradation within the samples. We compared species assignment results of twelve archaeological skin samples from Danish bogs using Mass Spectrometry (MS)-based peptide sequencing, against results obtained using light and scanning electron microscopy. While it was difficult to obtain reliable results using microscopy, MS enabled the identification of several species-diagnostic peptides, mostly from collagen and keratins, allowing confident species discrimination even among taxonomically close organisms, such as sheep and goat. Unlike previous MS-based methods, mostly relying on peptide fingerprinting, the shotgun sequencing approach we describe aims to identify the complete extracted ancient proteome, without preselected specific targets. As an example, we report the identification, in one of the samples, of two peptides uniquely assigned to bovine foetal haemoglobin, indicating the production of skin from a calf slaughtered within the first months of its life. We conclude that MS-based peptide sequencing is a reliable method for species identification of samples from bogs. The mass spectrometry proteomics data were deposited in the ProteomeXchange Consortium with the dataset identifier PXD001029. PMID:25260035

  13. Identification of novel Arabidopsis thaliana upstream open reading frames that control expression of the main coding sequences in a peptide sequence-dependent manner

    PubMed Central

    Ebina, Isao; Takemoto-Tsutsumi, Mariko; Watanabe, Shun; Koyama, Hiroaki; Endo, Yayoi; Kimata, Kaori; Igarashi, Takuya; Murakami, Karin; Kudo, Rin; Ohsumi, Arisa; Noh, Abdul Latif; Takahashi, Hiro; Naito, Satoshi; Onouchi, Hitoshi

    2015-01-01

    Upstream open reading frames (uORFs) are often found in the 5′-leader regions of eukaryotic mRNAs and can negatively modulate the translational efficiency of the downstream main ORF. Although the effects of most uORFs are thought to be independent of their encoded peptide sequences, certain uORFs control translation of the main ORF in a peptide sequence-dependent manner. For genome-wide identification of such peptide sequence-dependent regulatory uORFs, exhaustive searches for uORFs with conserved amino acid sequences have been conducted using bioinformatic analyses. However, whether the conserved uORFs identified by these bioinformatic approaches encode regulatory peptides has not been experimentally determined. Here we analyzed 16 recently identified Arabidopsis thaliana conserved uORFs for the effects of their amino acid sequences on the expression of the main ORF using a transient expression assay. We identified five novel uORFs that repress main ORF expression in a peptide sequence-dependent manner. Mutational analysis revealed that, in four of them, the C-terminal region of the uORF-encoded peptide is critical for the repression of main ORF expression. Intriguingly, we also identified one exceptional sequence-dependent regulatory uORF, in which the stop codon position is not conserved and the C-terminal region is not important for the repression of main ORF expression. PMID:25618853

  14. Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting.

    PubMed

    Tannu, Nilesh S; Wu, Jian; Rao, Vamshi K; Gadgil, Himanshu S; Pabst, Michael J; Gerling, Ivan C; Raghow, Rajendra

    2004-04-15

    We compared trysin-digested protein samples desalted by ZipTip(C18) reverse-phase microcolumns with on-plate washing of peptides deposited either on paraffin-coated plates (PCP), Teflon-based AnchorChip plates, or stainless steel plates, before analysis by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Trypsinized bovine serum albumin and ovalbumin and 16 protein spots extracted from silver-stained two-dimensional gels of murine C(2)C(12) myoblasts or human leukocytes, prepared by the above two methods, were subjected to MALDI on PCP, AnchorChip plates, or uncoated stainless steel plates. Although most peptide mass peaks were identical regardless of the method of desalting and concentrating of protein samples, samples washed and concentrated by the PCP-based method had peptide peaks that were not seen in the samples prepared using the ZipTip(C18) columns. The mass spectra of peptides desalted and washed on uncoated stainless steel MALDI plates were consistently inferior due to loss of peptides. Some peptides of large molecular masses were apparently lost from samples desalted by ZipTip(C18) microcolumns, thus diminishing the quality of the fingerprint needed for protein identification. We demonstrate that the method of washing of protein samples on paraffin-coated plates provides an easy, reproducible, inexpensive, and high-throughput alternative to ZipTip(C18)-based purification of protein prior to MALDI-TOF-MS analysis.

  15. Identification of New Snake Venom Metalloproteinase Inhibitors Using Compound Screening and Rational Peptide Design

    PubMed Central

    2012-01-01

    The majority of snakebite envenomations in Central America are caused by the viperid species Bothrops asper, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Broad metalloproteinase inhibitors, such as the peptidomimetic hydroxamate Batimastat, have been shown to inhibit snake venom metalloproteinases (SVMP). However, the difficulty in having open public access to Batimastat and similar molecules highlights the need to design new inhibitors of SVMPs that could be applied in the treatment of snakebite envenomations. We have chosen the SVMP BaP1 as a model to search for new inhibitors using different strategies, that is, screening of the Prestwick Chemical Library and rational peptide design. Results from these approaches provide clues on the structural requirements for efficient BaP1 inhibition and pave the way for the design of new inhibitors of SVMP. PMID:24900507

  16. Identification of new snake venom metalloproteinase inhibitors using compound screening and rational Peptide design.

    PubMed

    Villalta-Romero, Fabián; Gortat, Anna; Herrera, Andrés E; Arguedas, Rebeca; Quesada, Javier; de Melo, Robson Lopes; Calvete, Juan J; Montero, Mavis; Murillo, Renato; Rucavado, Alexandra; Gutiérrez, José María; Pérez-Payá, Enrique

    2012-07-12

    The majority of snakebite envenomations in Central America are caused by the viperid species Bothrops asper, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Broad metalloproteinase inhibitors, such as the peptidomimetic hydroxamate Batimastat, have been shown to inhibit snake venom metalloproteinases (SVMP). However, the difficulty in having open public access to Batimastat and similar molecules highlights the need to design new inhibitors of SVMPs that could be applied in the treatment of snakebite envenomations. We have chosen the SVMP BaP1 as a model to search for new inhibitors using different strategies, that is, screening of the Prestwick Chemical Library and rational peptide design. Results from these approaches provide clues on the structural requirements for efficient BaP1 inhibition and pave the way for the design of new inhibitors of SVMP. PMID:24900507

  17. Identification of an Orthogonal Peptide Binding Motif for Biarsenical Multiuse Affinity Probes

    SciTech Connect

    Chen, Baowei; Cao, Haishi; Yan, Ping; Mayer, M. Uljana; Squier, Thomas C.

    2007-07-01

    Biarsenical multiuse affinity probes (MAPs) complexed with ethanedithiol (EDT) permit the selective cellular labeling of proteins engineered with tetracysteine motifs, but are limited by the availability of a single binding motif (i.e., CCPGCC or PG tag) that prevents the differential labeling of co-expressed proteins. To overcome this problem, we have used a high-throughput peptide screen to identify an alternate binding motif (i.e., CCKACC or KA tag), which has a similar brightness to the classical sequence upon MAP binding, but displays altered rates and affinities of association that permit the differential labeling of these peptide sequences by the red probe 4,5-bis(1,3,2-dithiarsolan-2-yl)-resorufin (ReAsH-EDT2) or its green cognate 4’,5’-bis(1,3,2-dithoarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2 (FLAsH-EDT2). The utility of this labeling strategy was demonstrated following the expression of PG- and KA-tagged subunits of RNA polymerase expressed in E. coli. Specific labeling of two subunits of RNA polymerase in cellular lysates was achieved, whereby ReAsH-EDT2 is shown to selectively label the PG-tag on RNA polymerase alpha subunit prior to the labeling of the KA-tag sequence of the beta subunit of RNA polymerase with FlAsH-EDT2. These results demonstrate the ability to selectively label multiple individual proteins with orthogonal sequence tags in complex cellular lystates with spectroscopically distinct MAPs, and indicate the absolute specificity of ReAsH to target expressed proteins with essentially no nonspecific binding interactions.

  18. Identification of Symptomatic Fetuses Infected with Cytomegalovirus Using Amniotic Fluid Peptide Biomarkers

    PubMed Central

    Leruez-Ville, Marianne; Ramirez-Torres, Adela; Lacroix, Chrystelle; Breuil, Benjamin; Froment, Carine; Bascands, Jean-Loup; Schanstra, Joost P.; Ville, Yves

    2016-01-01

    Cytomegalovirus (CMV) is the most common cause of congenital infection, and is a major cause of sensorineural hearing loss and neurological disabilities. Evaluating the risk for a CMV infected fetus to develop severe clinical symptoms after birth is crucial to provide appropriate guidance to pregnant women who might have to consider termination of pregnancy or experimental prenatal medical therapies. However, establishing the prognosis before birth remains a challenge. This evaluation is currently based upon fetal imaging and fetal biological parameters, but the positive and negative predictive values of these parameters are not optimal, leaving room for the development of new prognostic factors. Here, we compared the amniotic fluid peptidome between asymptomatic fetuses who were born as asymptomatic neonates and symptomatic fetuses who were either terminated in view of severe cerebral lesions or born as severely symptomatic neonates. This comparison allowed us to identify a 34-peptide classifier in a discovery cohort of 13 symptomatic and 13 asymptomatic neonates. This classifier further yielded 89% sensitivity, 75% specificity and an area under the curve of 0.90 to segregate 9 severely symptomatic from 12 asymptomatic neonates in a validation cohort, showing an overall better performance than that of classical fetal laboratory parameters. Pathway analysis of the 34 peptides underlined the role of viral entry in fetuses with severe brain disease as well as the potential importance of both beta-2-microglobulin and adiponectin to protect the injured fetal brain infected with CMV. The results also suggested the mechanistic implication of the T calcium channel alpha-1G (CACNA1G) protein in the development of seizures in severely CMV infected children. These results open a new field for potential therapeutic options. In conclusion, this study demonstrates that amniotic fluid peptidome analysis can effectively predict the severity of congenital CMV infection. This

  19. Identification and functional characterization of an uncharacterized antimicrobial peptide from a ciliate Paramecium caudatum.

    PubMed

    Cui, Pengfei; Dong, Yuan; Li, Zhijian; Zhang, Yubo; Zhang, Shicui

    2016-07-01

    The global ever-growing concerns about multi-drug resistant (MDR) microbes leads to urgent demands for exploration of new antibiotics including antimicrobial peptides (AMPs). Here we demonstrated that a cDNA from Ciliata Paramecium caudatum, designated Pcamp1, coded for a protein with features characteristic of AMPs, which is not homologous to any AMPs currently known. Both the C-terminal 91 amino acid residues of PcAMP1, cPcAMP1, expressed in Escherichia coli and the C-terminal 26 amino acid residues (predicted mature AMP), cPcAMP1/26, synthesized, underwent a coil-to-helix transition in the presence of TFE, SDS or DPC. Functional assays revealed that cPcAMP1 and cPcAMP1/26 were both able to kill Aeromonas hydrophila and Staphylococcus aureus. ELISA showed that cPcAMP1 and cPcAMP1/26 were able to bind to microbe-associated molecular pattern molecules LPS and LTA, which was further corroborated by the observations that cPcAMP1 could deposit onto the bacterial membranes. Importantly, both cPcAMP1 and cPcAMP1/26 were able to induce bacterial membrane permeabilization and depolarization, and to increase intracellular ROS levels. Additionally, cPcAMP1 and cPcAMP1/26 were not cytotoxic to mammalian cells. Taken together, our results show that PcAMP1 is a potential AMP with a membrane selectivity towards bacterial cells, which renders it a promising template for the design of novel peptide antibiotics against MDR microbes. It also shows that use of signal conserved sequence of AMPs can be an effective tool to identify potential AMPs across different animal classes. PMID:26883426

  20. Identification of peptides that bind hepatitis C virus envelope protein E2 and inhibit viral cellular entry from a phage-display peptide library.

    PubMed

    Lü, Xin; Yao, Min; Zhang, Jian-Min; Yang, Jing; Lei, Ying-Feng; Huang, Xiao-Jun; Jia, Zhan-Sheng; Ma, Li; Lan, Hai-Yun; Xu, Zhi-Kai; Yin, Wen

    2014-05-01

    Hepatitis C virus (HCV) envelope protein E2 is required for the entry of HCV into cells. Viral envelope proteins interact with cell receptors in a multistep process, which may be a promising target for the development of novel antiviral agents. In this study, a heptapeptide M13 phage-display library was screened for peptides that bind specifically to prokaryotically expressed, purified truncated HCV envelope protein E2. ELISA assay was used to quantify the binding of the peptides to HCV E2 protein. Flow cytometry, quantitative reverse-transcription PCR and western blotting were used to investigate the inhibition effect of one peptide on HCV infection in hepatoma cells (Huh7.5) in vitro. Four peptides capable of binding specifically to HCV E2 protein were obtained after three rounds of biopanning. Peptide C18 (WPWHNHR), with the highest affinity for binding HCV E2 protein, was synthesized. The results showed that peptide C18 inhibited the viral infectivity of both HCV pseudotype particles (HCVpp) harboring HCV envelope glycoproteins and cell-culture produced HCV (HCVcc). Thus, this study demonstrated that peptide C18 is a potential candidate for anti-HCV therapy as a novel viral entry inhibitor.

  1. Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides.

    PubMed

    Chalkley, Robert J; Thalhammer, Agnes; Schoepfer, Ralf; Burlingame, A L

    2009-06-01

    Protein O-GlcNAcylation occurs in all animals and plants and is implicated in modulation of a wide range of cytosolic and nuclear protein functions, including gene silencing, nutrient and stress sensing, phosphorylation signaling, and diseases such as diabetes and Alzheimer's. The limiting factor impeding rapid progress in deciphering the biological functions of protein O-GlcNAcylation has been the inability to easily identify exact residues of modification. We describe a robust, high-sensitivity strategy able to assign O-GlcNAcylation sites of native modified peptides using electron transfer dissociation mass spectrometry. We have studied the murine postsynaptic density pseudoorganelle and report the assignment of 58 modification sites from a single experiment--significantly increasing the number of sites known in the literature. Components of several repressor complexes, such as NCoR1, polyhomeotic-like protein3, and EMSY, are modified. In addition, 28 O-GlcNAc sites were found on the protein Bassoon, effectively matching the number of phosphorylation sites reported previously on this protein. This finding suggests that on certain proteins, O-GlcNAcylation may be as extensive and important as phosphorylation in regulating protein function. Three of the newly discovered O-GlcNAc sites on Bassoon have previously been reported as phosphorylation sites, highlighting the interplay of the modifications. Surprisingly, several peptides with GlcNAc modifications on asparagines within the N-X-S/T consensus sequence were also observed from membrane protein extracellular domains. This powerful strategy fulfills a long-standing need in the biological community by facilitating modification site identifications that will accelerate understanding of the biological significance of this elusive regulatory posttranslational modification.

  2. Identification of novel helper epitope peptides of Survivin cancer-associated antigen applicable to developing helper/killer-hybrid epitope long peptide cancer vaccine.

    PubMed

    Ohtake, Junya; Ohkuri, Takayuki; Togashi, Yuji; Kitamura, Hidemitsu; Okuno, Kiyotaka; Nishimura, Takashi

    2014-09-01

    We identified novel helper epitope peptides of Survivin cancer antigen, which are presented to both HLA-DRB1*01:01 and DQB1*06:01. The helper epitope also contained three distinct Survivin-killer epitopes presented to HLA-A*02:01 and A*24:02. This 19 amino-acids epitope peptide (SU18) induced weak responses of Survivin-specific CD4(+) and CD8(+) T cells though it contained both helper and killer epitopes. To enhance the vaccine efficacy, we synthesized a long peptide by conjugating SU18 peptide and another DR53-restricted helper epitope peptide (SU22; 12 amino-acids) using glycine-linker. We designated this artificial 40 amino-acids long peptide containing two helper and three killer epitopes as Survivin-helper/killer-hybrid epitope long peptide (Survivin-H/K-HELP). Survivin-H/K-HELP allowed superior activation of IFN-γ-producing CD4(+) Th1 cells and CD8(+) Tc1 cells compared with the mixture of its component peptides (SU18 and SU22) in the presence of OK-432-treated monocyte-derived DC (Mo-DC). Survivin-H/K-HELP-pulsed Mo-DC pretreated with OK-432 also exhibited sustained antigen-presentation capability of stimulating Survivin-specific Th1 cells compared with Mo-DC pulsed with a mixture of SU18 and SU22 short peptides. Moreover, we demonstrated that Survivin-H/K-HELP induced a complete response in a breast cancer patient with the induction of cellular and humoral immune responses. Thus, we believe that an artificially synthesized Survivin-H/K-HELP will become an innovative cancer vaccine.

  3. Anti-coreceptor antibodies profoundly affect staining with peptide-MHC class I and class II tetramers.

    PubMed

    Wooldridge, Linda; Scriba, Thomas J; Milicic, Anita; Laugel, Bruno; Gostick, Emma; Price, David A; Phillips, Rodney E; Sewell, Andrew K

    2006-07-01

    The T cell coreceptors CD8 and CD4 bind to invariable regions of peptide-MHC class I (pMHCI) and class II (pMHCII) molecules, respectively, and facilitate antigen recognition by a number of mechanisms. It is established that some antibodies (Ab) specific for the CD8 molecule, which stabilizes TCR/pMHCI interactions, can alter the binding of pMHCI tetramers to cell surface TCR. In contrast, the extremely weak pMHCII/CD4 interaction does not stabilize TCR/pMHCII interactions or contribute to cognate tetramer binding; consequently, it is assumed that anti-CD4 Ab do not affect pMHCII binding. Here, we used a panel of point-mutated HLA A2 molecules with a range of affinities for CD8 spanning over three orders of magnitude to demonstrate that anti-CD8 Ab-mediated inhibition of pMHCI tetramer binding and cognate T cell activation correlates directly with the strength of the pMHCI/CD8 interaction. Further, some anti-CD4 Ab were found to block pMHCII tetramer binding; these effects were also paralleled in T cell activation assays. In sum, these data challenge the assertion that anti-coreceptor Ab exert their effects on T cell activation and pMHC binding solely by blocking pMHC/coreceptor interactions.

  4. Milk peptides increase iron solubility in water but do not affect DMT-1 expression in Caco-2 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In vitro digestion of milk produces peptide fractions that enhance iron uptake by Caco-2 cells. Our objectives were to investigate whether these fractions a) exert their effect by increasing relative gene expression of DMT-1 in Caco-2 cells b) enhance iron dialyzability when added in meals. Peptid...

  5. Ligand and Structure-Based Approaches for the Identification of Peptide Deformylase Inhibitors as Antibacterial Drugs

    PubMed Central

    Gao, Jian; Liang, Li; Zhu, Yasheng; Qiu, Shengzhi; Wang, Tao; Zhang, Ling

    2016-01-01

    Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins, which makes it an important antibacterial drug target. Given the importance of PDF inhibitors like actinonin in antibacterial drug discovery, several reported potent PDF inhibitors were used to develop pharmacophore models using the Galahad module of Sybyl 7.1 software. Generated pharmacophore models were composed of two donor atom centers, four acceptor atom centers and two hydrophobic groups. Model-1 was screened against the Zinc database and several compounds were retrieved as hits. Compounds with Qfit values of more than 60 were employed to perform a molecular docking study with the receptor Escherichia coli PDF, then compounds with docking score values of more than 6 were used to predict the in silico pharmacokinetic and toxicity risk via OSIRIS property explorer. Two known PDF inhibitors were also used to perform a molecular docking study with E. coli PDF as reference molecules. The results of the molecular docking study were validated by reproducing the crystal structure of actinonin. Molecular docking and in silico pharmacokinetic and toxicity prediction studies suggested that ZINC08740166 has a relatively high docking score of 7.44 and a drug score of 0.78. PMID:27428963

  6. Identification of novel serum peptide biomarkers for high-altitude adaptation: a comparative approach

    NASA Astrophysics Data System (ADS)

    Yang, Juan; Li, Wenhua; Liu, Siyuan; Yuan, Dongya; Guo, Yijiao; Jia, Cheng; Song, Tusheng; Huang, Chen

    2016-05-01

    We aimed to identify serum biomarkers for screening individuals who could adapt to high-altitude hypoxia at sea level. HHA (high-altitude hypoxia acclimated; n = 48) and HHI (high-altitude hypoxia illness; n = 48) groups were distinguished at high altitude, routine blood tests were performed for both groups at high altitude and at sea level. Serum biomarkers were identified by comparing serum peptidome profiling between HHI and HHA groups collected at sea level. Routine blood tests revealed the concentration of hemoglobin and red blood cells were significantly higher in HHI than in HHA at high altitude. Serum peptidome profiling showed that ten significantly differentially expressed peaks between HHA and HHI at sea level. Three potential serum peptide peaks (m/z values: 1061.91, 1088.33, 4057.63) were further sequence identified as regions of the inter-α trypsin inhibitor heavy chain H4 fragment (ITIH4 347–356), regions of the inter-α trypsin inhibitor heavy chain H1 fragment (ITIH1 205–214), and isoform 1 of fibrinogen α chain precursor (FGA 588–624). Expression of their full proteins was also tested by ELISA in HHA and HHI samples collected at sea level. Our study provided a novel approach for identifying potential biomarkers for screening people at sea level who can adapt to high altitudes.

  7. Identification of a Possible Role for Atrial Natiuretic Peptide in MDMA-induced hyperthermia

    PubMed Central

    Hrometz, Sandra L; Thatcher, Karen E; Ebert, Jeremy A; Mills, Edward M; Sprague, Jon E

    2011-01-01

    MDMA (3,4-methylenedioxymethamphetamine) induces thermogenesis in a mitochondrial uncoupling protein 3-dependent manner. There is evidence that this hyperthermia is mediated in part by the lipolytic release of free fatty acids, that subsequently activate uncoupling protein 3 in skeletal muscle mitochondria. We hypothesize that atrial natriuretic peptide (ANP), a strong lipolytic mediator, may contribute to the induction and maintenance of MDMA-induced thermogenesis. The specific aims of this study were to 1) determine if ANP is released following MDMA administration, and 2) use the ANP receptor antagonist, Anantin, to ascertain the role of ANP in MDMA-induced hyperthermia. ANP levels were measured in plasma at baseline, 10, 20, 30 and 60 min following MDMA (40 mg/kg, sc) administration in 16 male Sprague-Dawley rats. A robust increase in ANP was seen within ten min of MDMA administration. ANP levels returned to baseline at 20 min and then gradually rose over the 60 min monitoring period. The administration of Anantin (40 mg, ip), 15 min before and after MDMA, significantly attenuated the MDMA-induced hyperthermia. We conclude that ANP signaling contributes to the hyperthermia induced by MDMA. PMID:21827841

  8. Identification of TAX2 peptide as a new unpredicted anti-cancer agent.

    PubMed

    Jeanne, Albin; Sick, Emilie; Devy, Jérôme; Floquet, Nicolas; Belloy, Nicolas; Theret, Louis; Boulagnon-Rombi, Camille; Diebold, Marie-Danièle; Dauchez, Manuel; Martiny, Laurent; Schneider, Christophe; Dedieu, Stéphane

    2015-07-20

    The multi-modular glycoprotein thrombospondin-1 (TSP-1) is considered as a key actor within the tumor microenvironment. Besides, TSP-1 binding to CD47 is widely reported to regulate cardiovascular function as it promotes vasoconstriction and angiogenesis limitation. Therefore, many studies focused on targeting TSP-1:CD47 interaction, aiming for up-regulation of physiological angiogenesis to enhance post-ischemia recovery or to facilitate engraftment. Thus, we sought to identify an innovative selective antagonist for TSP-1:CD47 interaction. Protein-protein docking and molecular dynamics simulations were conducted to design a novel CD47-derived peptide, called TAX2. TAX2 binds TSP-1 to prevent TSP-1:CD47 interaction, as revealed by ELISA and co-immunoprecipitation experiments. Unexpectedly, TAX2 inhibits in vitro and ex vivo angiogenesis features in a TSP-1-dependent manner. Consistently, our data highlighted that TAX2 promotes TSP-1 binding to CD36-containing complexes, leading to disruption of VEGFR2 activation and downstream NO signaling. Such unpredicted results prompted us to investigate TAX2 potential in tumor pathology. A multimodal imaging approach was conducted combining histopathological staining, MVD, MRI analysis and μCT monitoring for tumor angiography longitudinal follow-up and 3D quantification. TAX2 in vivo administrations highly disturb syngeneic melanoma tumor vascularization inducing extensive tumor necrosis and strongly inhibit growth rate and vascularization of human pancreatic carcinoma xenografts in nude mice. PMID:26046793

  9. Identification of TAX2 peptide as a new unpredicted anti-cancer agent

    PubMed Central

    Jeanne, Albin; Sick, Emilie; Devy, Jérôme; Floquet, Nicolas; Belloy, Nicolas; Theret, Louis; Boulagnon-Rombi, Camille; Diebold, Marie-Danièle; Dauchez, Manuel; Martiny, Laurent; Schneider, Christophe; Dedieu, Stéphane

    2015-01-01

    The multi-modular glycoprotein thrombospondin-1 (TSP-1) is considered as a key actor within the tumor microenvironment. Besides, TSP-1 binding to CD47 is widely reported to regulate cardiovascular function as it promotes vasoconstriction and angiogenesis limitation. Therefore, many studies focused on targeting TSP-1:CD47 interaction, aiming for up-regulation of physiological angiogenesis to enhance post-ischemia recovery or to facilitate engraftment. Thus, we sought to identify an innovative selective antagonist for TSP-1:CD47 interaction. Protein-protein docking and molecular dynamics simulations were conducted to design a novel CD47-derived peptide, called TAX2. TAX2 binds TSP-1 to prevent TSP-1:CD47 interaction, as revealed by ELISA and co-immunoprecipitation experiments. Unexpectedly, TAX2 inhibits in vitro and ex vivo angiogenesis features in a TSP-1-dependent manner. Consistently, our data highlighted that TAX2 promotes TSP-1 binding to CD36-containing complexes, leading to disruption of VEGFR2 activation and downstream NO signaling. Such unpredicted results prompted us to investigate TAX2 potential in tumor pathology. A multimodal imaging approach was conducted combining histopathological staining, MVD, MRI analysis and μCT monitoring for tumor angiography longitudinal follow-up and 3D quantification. TAX2 in vivo administrations highly disturb syngeneic melanoma tumor vascularization inducing extensive tumor necrosis and strongly inhibit growth rate and vascularization of human pancreatic carcinoma xenografts in nude mice. PMID:26046793

  10. Identification of a β-glucosidase from the Mucor circinelloides genome by peptide pattern recognition.

    PubMed

    Huang, Yuhong; Busk, Peter Kamp; Grell, Morten Nedergaard; Zhao, Hai; Lange, Lene

    2014-12-01

    Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73U/mg) at pH5 and 50°C. The Km and Vmax with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20mM and 2.41U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation.

  11. Ligand and Structure-Based Approaches for the Identification of Peptide Deformylase Inhibitors as Antibacterial Drugs.

    PubMed

    Gao, Jian; Liang, Li; Zhu, Yasheng; Qiu, Shengzhi; Wang, Tao; Zhang, Ling

    2016-01-01

    Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins, which makes it an important antibacterial drug target. Given the importance of PDF inhibitors like actinonin in antibacterial drug discovery, several reported potent PDF inhibitors were used to develop pharmacophore models using the Galahad module of Sybyl 7.1 software. Generated pharmacophore models were composed of two donor atom centers, four acceptor atom centers and two hydrophobic groups. Model-1 was screened against the Zinc database and several compounds were retrieved as hits. Compounds with Qfit values of more than 60 were employed to perform a molecular docking study with the receptor Escherichia coli PDF, then compounds with docking score values of more than 6 were used to predict the in silico pharmacokinetic and toxicity risk via OSIRIS property explorer. Two known PDF inhibitors were also used to perform a molecular docking study with E. coli PDF as reference molecules. The results of the molecular docking study were validated by reproducing the crystal structure of actinonin. Molecular docking and in silico pharmacokinetic and toxicity prediction studies suggested that ZINC08740166 has a relatively high docking score of 7.44 and a drug score of 0.78. PMID:27428963

  12. Identification of novel serum peptide biomarkers for high-altitude adaptation: a comparative approach.

    PubMed

    Yang, Juan; Li, Wenhua; Liu, Siyuan; Yuan, Dongya; Guo, Yijiao; Jia, Cheng; Song, Tusheng; Huang, Chen

    2016-01-01

    We aimed to identify serum biomarkers for screening individuals who could adapt to high-altitude hypoxia at sea level. HHA (high-altitude hypoxia acclimated; n = 48) and HHI (high-altitude hypoxia illness; n = 48) groups were distinguished at high altitude, routine blood tests were performed for both groups at high altitude and at sea level. Serum biomarkers were identified by comparing serum peptidome profiling between HHI and HHA groups collected at sea level. Routine blood tests revealed the concentration of hemoglobin and red blood cells were significantly higher in HHI than in HHA at high altitude. Serum peptidome profiling showed that ten significantly differentially expressed peaks between HHA and HHI at sea level. Three potential serum peptide peaks (m/z values: 1061.91, 1088.33, 4057.63) were further sequence identified as regions of the inter-α trypsin inhibitor heavy chain H4 fragment (ITIH4 347-356), regions of the inter-α trypsin inhibitor heavy chain H1 fragment (ITIH1 205-214), and isoform 1 of fibrinogen α chain precursor (FGA 588-624). Expression of their full proteins was also tested by ELISA in HHA and HHI samples collected at sea level. Our study provided a novel approach for identifying potential biomarkers for screening people at sea level who can adapt to high altitudes.

  13. Identification of pregnancy-associated glycoproteins by peptide mass fingerprinting in water buffalo (Bubalus bubalis).

    PubMed

    Kumar, Pradeep; Saxena, Abhishake; Singh, S K; Sharma, R K; Singh, I; Agarwal, S K

    2014-08-01

    Ruminant placentas synthesize pregnancy-associated glycoproteins (PAGs) during pregnancy, which serve as biomarkers of pregnancy. The present study was conducted to verify, whether PAGs are expressed in buffalo placenta by using lectin-based affinity chromatography and peptide mass finger printing (PMF). Fetal cotyledonary tissues were collected from gravid uteri procured from slaughtered house. Proteins were extracted and subjected to wheat germ agglutinin (WGA) lectin affinity chromatography to isolate the PAGs. The isolated glycoproteins were separated by one-dimensional SDS-PAGE. PMF results of the 75 kDa protein revealed presence of two PAGs (PAG-7 and -11). The PAG-7 consisted of about 170 mass signals, of which 16 were assigned to corresponding/translated cDNA sequences of buffalo PAG-7, leading to sequence coverage of 40%. PMF result of PAG-11 showed 170 mass signals, of which 15 were assigned to buffalo PAG-11, leading to sequence coverage of 34%. In conclusion, the glycoprotein isolated from placental extract corresponding to 75 kDa band on SDS PAGE gel was a mixture of PAG-7 and -11, which may help in development of suitable diagnostics for pregnancy in buffalo.

  14. Identification and characterization of a novel prokaryotic peptide: N-glycosidase from Elizabethkingia meningoseptica.

    PubMed

    Sun, Guiqin; Yu, Xiang; Bao, Celimuge; Wang, Lei; Li, Meng; Gan, Jianhua; Qu, Di; Ma, Jinbiao; Chen, Li

    2015-03-20

    Peptide:N-glycosidase (PNGase) F, the first PNGase identified in prokaryotic cells, catalyzes the removal of intact asparagine-linked oligosaccharide chains from glycoproteins and/or glycopeptides. Since its discovery in 1984, PNGase F has remained as the sole prokaryotic PNGase. Recently, a novel gene encoding a protein with a predicted PNGase domain was identified from a clinical isolate of Elizabethkingia meningoseptica. In this study, the candidate protein was expressed in vitro and was subjected to biochemical and structural analyses. The results revealed that it possesses PNGase activity and has substrate specificity different from that of PNGase F. The crystal structure of the protein was determined at 1.9 Å resolution. Structural comparison with PNGase F revealed a relatively larger glycan-binding groove in the catalytic domain and an additional bowl-like domain with unknown function at the N terminus of the candidate protein. These structural and functional analyses indicated that the candidate protein is a novel prokaryotic N-glycosidase. The protein has been named PNGase F-II.

  15. The Protein Precursors of Peptides That Affect the Mechanics of Connective Tissue and/or Muscle in the Echinoderm Apostichopus japonicus

    PubMed Central

    Elphick, Maurice R.

    2012-01-01

    Peptides that cause muscle relaxation or contraction or that modulate electrically-induced muscle contraction have been discovered in the sea cucumber Apostichopus japonicus (Phylum Echinodermata; Class Holothuroidea). By analysing transcriptome sequence data, here the protein precursors of six of these myoactive peptides (the SALMFamides Sticho-MFamide-1 and -2, NGIWYamide, stichopin, GN-19 and GLRFA) have been identified, providing novel insights on neuropeptide and endocrine-type signalling systems in echinoderms. The A. japonicus SALMFamide precursor comprises eight putative neuropeptides including both L-type and F-type SALMFamides, which contrasts with previous findings from the sea urchin Strongylocentrotus purpuratus where L-type and F-type SALMFamides are encoded by different genes. The NGIWYamide precursor contains five copies of NGIWYamide but, unlike other NG peptide-type neuropeptide precursors in deuterostomian invertebrates, the NGIWYamide precursor does not have a C-terminal neurophysin domain, indicating loss of this character in holothurians. NGIWYamide was originally discovered as a muscle contractant, but it also causes stiffening of mutable connective tissue in the body wall of A. japonicus, whilst holokinins (PLGYMFR and derivative peptides) cause softening of the body wall. However, the mechanisms by which these peptides affect the stiffness of body wall connective tissue are unknown. Interestingly, analysis of the A. japonicus transcriptome reveals that the only protein containing the holokinin sequence PLGYMFR is an alpha-5 type collagen. This suggests that proteolysis of collagen may generate peptides (holokinins) that affect body wall stiffness in sea cucumbers, providing a novel perspective on mechanisms of mutable connective tissue in echinoderms. PMID:22952987

  16. The protein precursors of peptides that affect the mechanics of connective tissue and/or muscle in the echinoderm Apostichopus japonicus.

    PubMed

    Elphick, Maurice R

    2012-01-01

    Peptides that cause muscle relaxation or contraction or that modulate electrically-induced muscle contraction have been discovered in the sea cucumber Apostichopus japonicus (Phylum Echinodermata; Class Holothuroidea). By analysing transcriptome sequence data, here the protein precursors of six of these myoactive peptides (the SALMFamides Sticho-MFamide-1 and -2, NGIWYamide, stichopin, GN-19 and GLRFA) have been identified, providing novel insights on neuropeptide and endocrine-type signalling systems in echinoderms. The A. japonicus SALMFamide precursor comprises eight putative neuropeptides including both L-type and F-type SALMFamides, which contrasts with previous findings from the sea urchin Strongylocentrotus purpuratus where L-type and F-type SALMFamides are encoded by different genes. The NGIWYamide precursor contains five copies of NGIWYamide but, unlike other NG peptide-type neuropeptide precursors in deuterostomian invertebrates, the NGIWYamide precursor does not have a C-terminal neurophysin domain, indicating loss of this character in holothurians. NGIWYamide was originally discovered as a muscle contractant, but it also causes stiffening of mutable connective tissue in the body wall of A. japonicus, whilst holokinins (PLGYMFR and derivative peptides) cause softening of the body wall. However, the mechanisms by which these peptides affect the stiffness of body wall connective tissue are unknown. Interestingly, analysis of the A. japonicus transcriptome reveals that the only protein containing the holokinin sequence PLGYMFR is an alpha-5 type collagen. This suggests that proteolysis of collagen may generate peptides (holokinins) that affect body wall stiffness in sea cucumbers, providing a novel perspective on mechanisms of mutable connective tissue in echinoderms.

  17. Skin peptide and cDNA profiling of Australian anurans: genus and species identification and evolutionary trends.

    PubMed

    Jackway, Rebecca J; Pukala, Tara L; Donnellan, Stephen C; Sherman, Patrick J; Tyler, Michael J; Bowie, John H

    2011-01-01

    Host defense peptides of 35 species of Australian frogs from the hylids Cyclorana and Litoria, and the myobatrachids Crinia, Limnodynastes and Uperoleia have been identified. The biological activities of the majority of these peptides have been determined and include hormones, neuropeptides, opioids, immunomodulators, membrane active peptides [including antimicrobial, anticancer, antiviral (enveloped viruses like HIV and Herpes) and antifungal peptides], neuronal nitric oxide synthase inhibitors, pheromones and individual peptides with other specific activities. The host defense peptide skin profile can be diagnostic at both the species and higher taxonomic levels; for example, species of Crinia, Litoria and Uperoleia each produce quite different types of peptides. Species of Cyclorana and Limnodynastes are more difficult to characterize by skin peptides alone: species of both genera produce similar peptides with no apparent activity. The skin peptide profiles of frogs from the genera Crinia, Litoria and Uperoleia may be used together with morphological and cognate methods, to differentiate between sub-species and even different population clusters of the same species. Nucleotide sequencing of cDNAs of precursors (pre-pro peptides) of bioactive peptides from the skin glands of various species of the genus Litoria show that the majority of these peptides originated from a single ancestor gene before the break away of Australia from Gondwana. The exceptions are the caerulein neuropeptides {e.g. caerulein [pEQDY(SO(3)H)TGWMDF(NH(2))]} which have a different origin to that of other Litoria peptides. Disulfide containing peptides from skin glands of species of Crinia show a different evolutionary route to peptides from species of Litoria.

  18. Identification of biological pathways regulated by PGRN and GRN peptide treatments using transcriptome analysis.

    PubMed

    Rollinson, Sara; Young, Kate; Bennion-Callister, Janis; Pickering-Brown, Stuart M

    2016-09-01

    Mutations in progranulin (PGRN) have been linked to two neurodegenerative disorders, heterozygote mutations with frontotemporal lobar degeneration (FTLD) and homozygote mutations with neuronal ceroid lipofuscinosis (NCL). Human PGRN is 593aa secreted growth factor, made up of seven and a half repeats of a highly conserved granulin motif that is cleaved to produce the granulin peptides A-G and paragranulin. While it is thought that PGRN protects against neurodegeneration through its role in inflammation and tissue repair, the role of PGRN and granulins in the nervous system is currently unclear. To better understand this, we prepared recombinant PGRN, granulin A-F and paragranulin, and used these to treat differentiated neuronal SH-SY5Y cells. Using RNA sequencing and bioinformatics techniques we investigated the functional effects of PGRN and the individual granulins upon the transcriptome. For PGRN treatment we show that the main effect of short-duration treatments is the down-regulation of transcripts, supporting that signalling pathway induction appears to be dominant effect. Gene ontology analysis, however, also supports the regulation of biological processes such as the spliceosome and proteasome in response to PGRN treatment, as well as the lysosomal pathway constituents such as CHMP1A, further supporting the role of PGRN in lysosomal function. We also show that the response to granulin treatments involves the regulation of numerous non-coding RNA's, and the granulins cluster into groups of similar activity on the basis of expression profile with paragranulin and PGRN having similar expression profiles, while granulins B, D, E and G appear more similar. PMID:27307215

  19. Rapid Generation of a Nanocrystal-Labeled Peptide Library for Specific Identification of the Bacterium Clostrium Botulinum

    SciTech Connect

    Tok, J B

    2004-11-11

    Several peptide libraries containing up to 2 million unique peptide ligands have been synthesized. The peptides are attached onto a 80 micron resin and the length of these peptide ligands ranges from 5 to 9 amino acid residues. Using a novel calorimetric assay, the libraries were screened for binding to the ganglioside-binding domain of Clostridium Tetanus Toxin, a structural similar analog of the Clostridium Botulinum toxin. Several binding peptide sequences were identified, in which the detailed binding kinetics are currently underway using the Surface Plasmon Resonance (SPR) technique.

  20. Identification of peptides in human Hsp20 and Hsp27 that possess molecular chaperone and anti-apoptotic activities

    PubMed Central

    Nahomi, Rooban B.; DiMauro, Michael A.; Wang, Benlian; Nagaraj, Ram H.

    2015-01-01

    Previous studies have identified peptides in the ‘crystallin-domain’ of the small heat-shock protein (sHSP) α-crystallin with chaperone and anti-apoptotic activities. We found that peptides in heat-shock protein Hsp20 (G71HFSVLLDVKHFSPEEIAVK91) and Hsp27 (D93RWRVSLDVNHFAPDELTVK113) with sequence homology to α-crystallin also have robust chaperone and anti-apoptotic activities. Both peptides inhibited hyperthermic and chemically induced aggregation of client proteins. The scrambled peptides of Hsp20 and Hsp27 showed no such effects. The chaperone activities of the peptides were better than those from αA- and αB-crystallin. HeLa cells took up the FITC-conjugated Hsp20 peptide and, when the cells were thermally stressed, the peptide was translocated from the cytoplasm to the nucleus. The two peptides inhibited apoptosis in HeLa cells by blocking cytochrome c release from the mitochondria and caspase-3 activation. We found that scrambling the last four amino acids in the two peptides (KAIV in Hsp20 and KTLV in Hsp27) made them unable to enter cells and ineffective against stress-induced apoptosis. Intraperitoneal injection of the peptides prevented sodium-selenite-induced cataract formation in rats by inhibiting protein aggregation and oxidative stress. Our study has identified peptides from Hsp20 and Hsp27 that may have therapeutic benefit in diseases where protein aggregation and apoptosis are contributing factors. PMID:25332102

  1. Inhibition of verocytotoxigenic Escherichia coli by antimicrobial peptides caseicin A and B and the factors affecting their antimicrobial activities.

    PubMed

    McDonnell, Mary J; Rivas, Lucia; Burgess, Catherine M; Fanning, Séamus; Duffy, Geraldine

    2012-02-15

    The antimic robial activities of caseicin A and B antimicrobial peptides (AMPs) were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains (n=11), other bacterial pathogenic and spoilage bacteria (n=7), using a model broth system. The ability of the AMPs to retain their antimicrobial activities against a strain of E. coli O157:H7 380-94 under various test conditions (pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competitive microflora) was assessed and the minimum inhibitory concentrations (MIC) and number of surviving E. coli O157:H7 calculated. The mean number of VTEC surviving after exposure to 2 mg/ml caseicin A and B was reduced by 4.96 and 4.19 log(10) cfu/ml compared to the respective controls. The susceptibility of E. coli O157:H7 to the caseicin AMPs decreased as temperature, pH, water activity and inoculum size were reduced. The presence of sodium chloride (0.5-2.5%) did not affect the activity of caseicin A (p>0.05), however it did inhibit the activity of caseicin B. The presence of a competitive microflora cocktail did not significantly (p>0.05) affect the activities of the AMPs for the majority of the concentrations tested. Using a quantitative PCR assay, the levels of verotoxins (vt1 and vt2) expressed by E. coli O157:H7 following exposure to a sub-inhibitory concentration (0.5 mg/ml) of caseicin A showed that the verotoxin levels did not differ from the levels produced by the control cultures. The antimicrobial activity of caseicin A against E. coli O157:H7 was also tested in a model rumen system, however concentrations of ≥2 mg/ml did not significantly (p>0.05) reduce E. coli O157:H7 numbers in the model system over a 24 h period. The application of caseicin AMPs in food and/or animal production may be valuable in combination with other antimicrobials although further research is required.

  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. Species identification of Oetzi's clothing with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on peptide pattern similarities of hair digests.

    PubMed

    Hollemeyer, Klaus; Altmeyer, Wolfgang; Heinzle, Elmar; Pitra, Christian

    2008-09-01

    Identification of ancient biological samples from the 1991-discovered and more than 5300-year-old Tyrolean mummy, also called iceman or Oetzi, is very difficult. The species of origins of four animal-hair-bearing samples of the accoutrement of the mummy not yet diagnosed were identified by a special proteomics method. Ha 43/91/130 and Ha 6/91, two samples from his coat, and Ha 5/91, a sample from his leggings, were assigned to sheep. The upper leather of his moccasins, Ha 2/91, was made from cattle. Despite the enormous age of these samples with partial (bio)chemical alterations, reliable identification was possible using a recently developed matrix-assisted laser desorption/ionization time-of-flight mass spectrometric ((MALDI-TOF MS)-based analytical method. The method is exclusively based on the analysis of proteins and uses minute amounts of peptides directly derived from tryptic hair digests without any separation or enrichment steps. Unknown species are identified by comparison of their peptide ion patterns with known spectra stored in existing databases. Hereby, the correlation distance, a form of Euclidean distance, and deduced parameters are used to measure similarities. If more than one potential hit remains, specific diagnostic peptide ions are used to stepwise exclude incorrect matches. These ions are specific for orders, families, subfamilies/genera and/or even species. Peptide mass fingerprinting data combined with those from collision-induced dissociation spectra (combined MS & MS/MS) were used for interpretation with the MASCOT search engine and the NCBI database to find the potential parentage of hair proteins. For this technique, selected precursor ions were identified as specific diagnostic peptide ions.

  4. NOVEL CONTINUOUS PH/SALT GRADIENT AND PEPTIDE SCORE FOR STRONG CATION EXCHANGE CHROMATOGRAPHY IN 2D-NANO-LC/MSMS PEPTIDE IDENTIFICATION FOR PROTEOMICS

    EPA Science Inventory

    Tryptic digests of human serum albumin (HSA) and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MSMS with strong cation exchange (SCX) and reverse phase (RP) chromatography and contin...

  5. Post-glucose-load urinary C-peptide and glucose concentration obtained during OGTT do not affect oral minimal model-based plasma indices.

    PubMed

    Jainandunsing, Sjaam; Wattimena, J L Darcos; Rietveld, Trinet; van Miert, Joram N I; Sijbrands, Eric J G; de Rooij, Felix W M

    2016-05-01

    The purpose of this study was to investigate how renal loss of both C-peptide and glucose during oral glucose tolerance test (OGTT) relate to and affect plasma-derived oral minimal model (OMM) indices. All individuals were recruited during family screening between August 2007 and January 2011 and underwent a 3.5-h OGTT, collecting nine plasma samples and urine during OGTT. We obtained the following three subgroups: normoglycemic, at risk, and T2D. We recruited South Asian and Caucasian families, and we report separate analyses if differences occurred. Plasma glucose, insulin, and C-peptide concentrations were analyzed as AUCs during OGTT, OMM estimate of renal C-peptide secretion, and OMM beta-cell and insulin sensitivity indices were calculated to obtain disposition indices. Post-glucose load glucose and C-peptide in urine were measured and related to plasma-based indices. Urinary glucose corresponded well with plasma glucose AUC (Cau r = 0.64, P < 0.01; SA r = 0.69, P < 0.01), S I (Cau r = -0.51, P < 0.01; SA r = -0.41, P < 0.01), Φ dynamic (Cau r = -0.41, P < 0.01; SA r = -0.57, P < 0.01), and Φ oral (Cau r = -0.61, P < 0.01; SA r = -0.73, P < 0.01). Urinary C-peptide corresponded well to plasma C-peptide AUC (Cau r = 0.45, P < 0.01; SA r = 0.33, P < 0.05) and OMM estimate of renal C-peptide secretion (r = 0.42, P < 0.01). In general, glucose excretion plasma threshold for the presence of glucose in urine was ~10-10.5 mmol L(-1) in non-T2D individuals, but not measurable in T2D individuals. Renal glucose secretion during OGTT did not influence OMM indices in general nor in T2D patients (renal clearance range 0-2.1 %, with median 0.2 % of plasma glucose AUC). C-indices of urinary glucose to detect various stages of glucose intolerance were excellent (Cau 0.83-0.98; SA 0.75-0.89). The limited role of renal glucose secretion validates the neglecting of urinary glucose secretion in kinetic models of glucose homeostasis using plasma glucose concentrations. Both C-peptide

  6. Identification of protein complexes in Escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry.

    PubMed

    Babu, Mohan; Kagan, Olga; Guo, Hongbo; Greenblatt, Jack; Emili, Andrew

    2012-01-01

    Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems(1, 2). Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria(1-6). In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes(1, 2, 7). Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast(8, 9), and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system(10). Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large

  7. Factors and Trends Affecting the Identification of a Reliable Biomarker for Diesel Exhaust Exposure

    PubMed Central

    2014-01-01

    The monitoring of human exposures to diesel exhaust continues to be a vexing problem for specialists seeking information on the potential health effects of this ubiquitous combustion product. Exposure biomarkers have yielded a potential solution to this problem by providing a direct measure of an individual's contact with key components in the exhaust stream. Spurred by the advent of new, highly sensitive, analytical methods capable of detecting substances at very low levels, there have been numerous attempts at identifying a stable and specific biomarker. Despite these new techniques, there is currently no foolproof method for unambiguously separating diesel exhaust exposures from those arising from other combustion sources. Diesel exhaust is a highly complex mixture of solid, liquid, and gaseous components whose exact composition can be affected by many variables, including engine technology, fuel composition, operating conditions, and photochemical aging. These factors together with those related to exposure methodology, epidemiological necessity, and regulatory reform can have a decided impact on the success or failure of future research aimed at identifying a suitable biomarker of exposure. The objective of this review is to examine existing information on exposure biomarkers for diesel exhaust and to identify those factors and trends that have had an impact on the successful identification of metrics for both occupational and community settings. The information will provide interested parties with a template for more thoroughly understanding those factors affecting diesel exhaust emissions and for identifying those substances and research approaches holding the greatest promise for future success. PMID:25170242

  8. Identification of HLA-A24-binding peptides of Mycobacterium tuberculosis derived proteins with beta 2m linked HLA-A24 single chain expressing cells.

    PubMed

    Ding, Jie; Wang, Yan; Cheng, Tingting; Chen, Xiaowei; Gao, Bin

    2010-01-01

    Tuberculosis is caused by an intracellular pathogen Mycobacterium tuberculosis (Mtb) and poses a persistent threat to global health. MHC class I-restricted CD8 cytotoxic T lymphocytes (CTL) are essential for protective immunity to Tuberculosis. Information for CTL epitopes derived from Mtb is desirable for vaccine design and assessment of T cell responses. However, the knowledge about CTL epitopes of Mtb, particularly those non-A2 HLA alleles restricted is rare. In this study, beta-2-microglobulin (beta 2m, beta(2)m) linked HLA-A24 single chain was expressed on RMA-S cell line defective in the endogenous antigen processing and applied for screening of peptides which could stabilize the HLA-A24 complex on the cell surface. From a group of peptides predicted as binders by a computer algorithm, five peptides were shown to bind to HLA-A24 protein on the cell surface. As comparison we have also identified a dozen Mtb proteins derived peptides that bind to HLA-A2 specifically. The cell line and HLA binders present here would be useful for further identification of CD8 restricted Mtb epitopes.

  9. Family identification: a beneficial process for young adults who grow up in homes affected by parental intimate partner violence

    PubMed Central

    Naughton, Catherine M.; Muldoon, Orla T.

    2015-01-01

    Exposure to parental intimate partner violence (parental IPV) is a complex trauma. Research within social psychology establishes that identification with social groups impacts positively on how we appraise, respond to and recover from traumatic events. IPV is also a highly stigmatized social phenomenon and social isolation is a major factor for families affected by IPV, yet strong identification with the family group may act as a beneficial psychological resource to young people who grew up in homes affected by IPV. The current study, an online survey of 355 students (Mage = 20, 70% female), investigated if a psychosocial process, specifically identification with the family, may influence the relationship between the predictor, exposure to parental IPV, and outcomes, global self-esteem and state anxiety. Mediation analysis suggests that identification with the family has a positive influence on the relationship between exposure to parental IPV and psychological outcomes; exposure to parental IPV results in reduced family identification, but when family identification is strong it results in both reduced anxiety and increased self-esteem for young people. The findings highlight the importance of having a strong sense of belonging to the extended family for young people who were exposed to parental IPV, thus has implications for prevention, intervention, and social policy. PMID:26379582

  10. Family identification: a beneficial process for young adults who grow up in homes affected by parental intimate partner violence.

    PubMed

    Naughton, Catherine M; O'Donnell, Aisling T; Muldoon, Orla T

    2015-01-01

    Exposure to parental intimate partner violence (parental IPV) is a complex trauma. Research within social psychology establishes that identification with social groups impacts positively on how we appraise, respond to and recover from traumatic events. IPV is also a highly stigmatized social phenomenon and social isolation is a major factor for families affected by IPV, yet strong identification with the family group may act as a beneficial psychological resource to young people who grew up in homes affected by IPV. The current study, an online survey of 355 students (M age = 20, 70% female), investigated if a psychosocial process, specifically identification with the family, may influence the relationship between the predictor, exposure to parental IPV, and outcomes, global self-esteem and state anxiety. Mediation analysis suggests that identification with the family has a positive influence on the relationship between exposure to parental IPV and psychological outcomes; exposure to parental IPV results in reduced family identification, but when family identification is strong it results in both reduced anxiety and increased self-esteem for young people. The findings highlight the importance of having a strong sense of belonging to the extended family for young people who were exposed to parental IPV, thus has implications for prevention, intervention, and social policy.

  11. Family identification: a beneficial process for young adults who grow up in homes affected by parental intimate partner violence.

    PubMed

    Naughton, Catherine M; O'Donnell, Aisling T; Muldoon, Orla T

    2015-01-01

    Exposure to parental intimate partner violence (parental IPV) is a complex trauma. Research within social psychology establishes that identification with social groups impacts positively on how we appraise, respond to and recover from traumatic events. IPV is also a highly stigmatized social phenomenon and social isolation is a major factor for families affected by IPV, yet strong identification with the family group may act as a beneficial psychological resource to young people who grew up in homes affected by IPV. The current study, an online survey of 355 students (M age = 20, 70% female), investigated if a psychosocial process, specifically identification with the family, may influence the relationship between the predictor, exposure to parental IPV, and outcomes, global self-esteem and state anxiety. Mediation analysis suggests that identification with the family has a positive influence on the relationship between exposure to parental IPV and psychological outcomes; exposure to parental IPV results in reduced family identification, but when family identification is strong it results in both reduced anxiety and increased self-esteem for young people. The findings highlight the importance of having a strong sense of belonging to the extended family for young people who were exposed to parental IPV, thus has implications for prevention, intervention, and social policy. PMID:26379582

  12. A mutation in the alpha 3 domain of Db that abrogates CD8 binding does not affect presentation of an immunodominant H-Y peptide.

    PubMed Central

    Dutz, J P; Teh, S J; Killeen, N; Teh, H S

    1995-01-01

    The peptidic nature of the male (H-Y) antigen, a model minor histocompatibility antigen in H-2b mice, has recently been demonstrated. In this study we show that the H-Y peptide, which is recognized by PM-1, a Db-restricted cytotoxic T-lymphocyte (CTL) clone, is absent in male H-2d spleen cells but present in male H-2d spleen cells that also express a transgenic Db molecule under its endogenous promoter. This result indicates that both the H-Y and the Db gene products are essential and sufficient for production of the Db-restricted H-Y peptide. By comparing the ability of the PM-1 clone and bulk CTL generated in a secondary mixed lymphocyte culture to recognize H-Y peptidic material eluted from affinity-purified Db molecules and separated by reversed-phase high-performance liquid chromatography (HPLC), we provide evidence that there is an immunodominant H-Y epitope that is presented by the Db molecule. Furthermore, the presentation of this epitope is not affected by a mutation in the alpha 3 domain of Db (asp227 to lys227), which abrogates CD8 binding, since similar amounts of H-Y peptide were eluted from affinity-purified wild-type or mutant Db molecules. However, the generation of the H-Y epitope is dependent on the presence of beta 2-microglobulin, since it is absent in male H-2b mice that lack a functional beta 2-microglobulin gene. The implications of these findings on T-cell development are discussed. PMID:7543449

  13. Dutch and arctic mutant peptides of β amyloid1–40 differentially affect the FGF-2 pathway in brain endothelium

    PubMed Central

    Solito, Raffaella; Corti, Federico; Fossati, Silvia; Mezhericher, Emiliya; Donnini, Sandra; Ghiso, Jorge; Giachetti, Antonio; Rostagno, Agueda; Ziche, Marina

    2009-01-01

    Single point mutations of the amyloid precursor protein generate Aβ variants bearing amino acid substitutions at positions 21–23. These mutants are associated with distinct hereditary phenotypes of cerebral amyloid angiopathy, manifesting varying degrees of tropism for brain vessels, and impaired microvessel remodeling and angiogenesis. We examined the differential effects of E22Q (Dutch), and E22G (Arctic) variants in comparison to WT Aβ on brain endothelial cell proliferation, angiogenic phenotype expression triggered by fibroblast growth factor (FGF-2), pseudo-capillary sprouting, and induction of apoptosis. E22Q exhibited a potent anti-angiogenic profile in contrast to E22G, which had a much weaker effect. Investigations on the FGF-2 signaling pathway revealed the greatest differences among the peptides: E22Q andWT peptides suppressed FGF-2 expression while E22G had barely any effect. Phosphorylation of the FGF-2 receptor, FGFR-1, and the survival signal Akt were abolished by E22Q and WT peptides, but not by E22G. The biological dissimilar effect of the mutant and WT peptides on cerebral EC cannot be assigned to a particular Aβ structure, suggesting that the toxic effect of the Aβ assemblies goes beyond mere multimerization. PMID:19061884

  14. Zn(II) chelating with peptides found in sesame protein hydrolysates: identification of the binding sites of complexes.

    PubMed

    Wang, Chan; Wang, Chan; Li, Bo; Li, Haixia

    2014-12-15

    Two metal-chelating peptides identified from sesame protein hydrolysates, Ser-Met (SM) and Asn-Cys-Ser (NCS) were chemically synthesized in order to study zinc-peptide complexes. The stability constants of two peptides and their zinc complexations were measured by pH-potentiometric techniques, and the molecular masses of the Zn-peptide complexes were determined by electrospray ionization mass spectrometry. The structures of the complexes were studied by infrared spectroscopy and quantum chemistry and possible conformations of the Zn-peptide complexes were determined by molecular modeling to obtain direct insights into the molecular mechanism of Zn(2+) chelation with peptides. The results confirmed that the zinc ion-to-ligand ratio is 1:1 for the two peptides and that water is involved in the formation of the complexes. Among the functional groups of SM and NCS, the carboxyl, hydroxyl, and sulfhydryl groups showed the strongest bonding abilities with Zn(2+) and the carbonyl group of the peptide bond and water (O) regularly participate in coordination by weaker interactions with Zn(2+).

  15. Identification and Characterization of a Small Inhibitory Peptide That Can Target DNA-PKcs Autophosphorylation and Increase Tumor Radiosensitivity

    SciTech Connect

    Sun Xiaonan; Yang Chunying; Liu Hai; Wang Qi; Wu Shixiu; Li Xia; Xie Tian; Brinkman, Kathryn L.; Teh, Bin S.; Butler, E. Brian; Xu Bo; Zheng, Shu

    2012-12-01

    Purpose: The DNA protein kinase catalytic subunit (DNA-PKcs) is one of the critical elements involved in the DNA damage repair process. Inhibition of DNA-PKcs results in hypersensitivity to ionizing radiation (IR); therefore, this approach has been explored to develop molecular targeted radiosensitizers. Here, we aimed to develop small inhibitory peptides that could specifically target DNA-PKcs autophosphorylation, a critical step for the enzymatic activation of the kinase in response to IR. Methods and Materials: We generated several small fusion peptides consisting of 2 functional domains, 1 an internalization domain and the other a DNA-PKcs autophosphorylation inhibitory domain. We characterized the internalization, toxicity, and radiosensitization activities of the fusion peptides. Furthermore, we studied the mechanisms of the inhibitory peptides on DNA-PKcs autophosphorylation and DNA repair. Results: We found that among several peptides, the biotin-labeled peptide 3 (BTW3) peptide, which targets DNA-PKcs threonine 2647 autophosphorylation, can abrogate IR-induced DNA-PKcs activation and cause prolonged {gamma}-H2AX focus formation. We demonstrated that BTW3 exposure led to hypersensitivity to IR in DNA-PKcs-proficient cells but not in DNA-PKcs-deficient cells. Conclusions: The small inhibitory peptide BTW3 can specifically target DNA-PKcs autophosphorylation and enhance radiosensitivity; therefore, it can be further developed as a novel class of radiosensitizer.

  16. Identification and characterization of a sex peptide receptor-like transcript from the western tarnished plant bug, Lygus hesperus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lygus hesperus females exhibit a post-mating behavioral switch that triggers increased egg laying and decreased sexual interest. In Drosophila melanogaster, post-mating changes in behavior are controlled by sex peptide (SP) and the sex peptide receptor (DmSPR). SPR is present in most insect genome...

  17. Identification of Antihypertensive Peptides Derived from Low Molecular Weight Casein Hydrolysates Generated during Fermentation by Bifidobacterium longum KACC 91563

    PubMed Central

    Ha, Go Eun; Chang, Oun Ki; Jo, Su-Mi; Han, Gi-Sung; Park, Beom-Young; Ham, Jun-Sang; Jeong, Seok-Geun

    2015-01-01

    Angiotensin-converting enzyme (ACE) inhibitory activity was evaluated for the low-molecular-weight fraction (<3 kDa) obtained from milk fermentation by Bifidobacterium longum KACC91563. The ACE inhibitory activity in this fraction was 62.3%. The peptides generated from the <3 kDa fraction were identified by liquid chromatography-electrospray ionization quantitative time-of-flight mass spectrometry analysis. Of the 28 peptides identified, 11 and 16 were identified as β-casein (CN) and αs1-CN, respectively. One peptide was identified as κ-CN. Three peptides, YQEPVLGPVRGPFPIIV, QEPVLGPVRGPFPIIV, and GPVRGPFPIIV, from β-CN corresponded to known antihypertensive peptides. We also found 15 peptides that were identified as potential antihypertensive peptides because they included a known antihypertensive peptide fragment. These peptides were as follows: RELEELNVPGEIVE (f1-14), YQEPVLGPVRGPFP (f193-206), EPVLGPVRGPFPIIV (f195-206), PVLGPVRGPFPIIV (f196-206), VLGPVRGPFPIIV (f197-206), and LGPVRGPFPIIV (f198-206) for β-CN; and APSFSDIPNPIGSENSEKTTMPLW (f176-199), SFSDIPNPIGSENSEKT- TMPLW (f178-199), FSDIPNPIGSENSEKTTMPLW (f179-199), SDIPNPIGSENSEKTTMPLW (f180-199), DIPNPIGSENSEKTTMPLW (f181-199), IPNPIGSENSEKTTMPLW (f182-199), PIGSENSEKTTMPLW (f185-199), IGSENSEKTTMPLW (f186-199), and SENSEKTTMPLW (f188-199) for αs1-CN. From these results, B. longum could be used as a starter culture in combination with other lactic acid bacteria in the dairy industry, and/or these peptides could be used in functional food manufacturing as additives for the development of a product with beneficial effects for human health. PMID:26877633

  18. The peptide semax affects the expression of genes related to the immune and vascular systems in rat brain focal ischemia: genome-wide transcriptional analysis

    PubMed Central

    2014-01-01

    Background The nootropic neuroprotective peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has proved efficient in the therapy of brain stroke; however, the molecular mechanisms underlying its action remain obscure. Our genome-wide study was designed to investigate the response of the transcriptome of ischemized rat brain cortex tissues to the action of Semax in vivo. Results The gene-expression alteration caused by the action of the peptide Semax was compared with the gene expression of the “ischemia” group animals at 3 and 24 h after permanent middle cerebral artery occlusion (pMCAO). The peptide predominantly enhanced the expression of genes related to the immune system. Three hours after pMCAO, Semax influenced the expression of some genes that affect the activity of immune cells, and, 24 h after pMCAO, the action of Semax on the immune response increased considerably. The genes implicated in this response represented over 50% of the total number of genes that exhibited Semax-induced altered expression. Among the immune-response genes, the expression of which was modulated by Semax, genes that encode immunoglobulins and chemokines formed the most notable groups. In response to Semax administration, 24 genes related to the vascular system exhibited altered expression 3 h after pMCAO, whereas 12 genes were changed 24 h after pMCAO. These genes are associated with such processes as the development and migration of endothelial tissue, the migration of smooth muscle cells, hematopoiesis, and vasculogenesis. Conclusions Semax affects several biological processes involved in the function of various systems. The immune response is the process most markedly affected by the drug. Semax altered the expression of genes that modulate the amount and mobility of immune cells and enhanced the expression of genes that encode chemokines and immunoglobulins. In conditions of rat brain focal ischemia, Semax influenced the expression of genes that promote the formation and

  19. Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers

    SciTech Connect

    James Graham, Robert Leslie . E-mail: rl.graham@ulster.ac.uk; Graham, Ciaren; McClean, Stephen; Chen, Tianbao; O'Rourke, Martin; Hirst, David; Theakston, David; Shaw, Chris

    2005-12-23

    A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.

  20. Identification and Tumour-Binding Properties of a Peptide with High Affinity to the Disialoganglioside GD2

    PubMed Central

    Müller, Jan; Reichel, Robin; Vogt, Sebastian; Müller, Stefan P.; Sauerwein, Wolfgang; Brandau, Wolfgang; Eggert, Angelika

    2016-01-01

    Neuroectodermal tumours are characterized by aberrant processing of disialogangliosides concomitant with high expression of GD2 or GD3 on cell surfaces. Antibodies targeting GD2 are already in clinical use for therapy of neuroblastoma, a solid tumour of early childhood. Here, we set out to identify peptides with high affinity to human disialoganglioside GD2. To this end, we performed a combined in vivo and in vitro screen using a recombinant phage displayed peptide library. We isolated a phage displaying the peptide sequence WHWRLPS that specifically binds to the human disialoganglioside GD2. Binding specificity was confirmed by mutational scanning and by comparative analyses using structurally related disialogangliosides. In vivo, significant enrichment of phage binding to xenografts of human neuroblastoma cells in mice was observed. Tumour-specific phage accumulation could be blocked by intravenous coinjection of the corresponding peptide. Comparative pharmacokinetic analyses revealed higher specific accumulation of 68Ga-labelled GD2-binding peptide compared to 111In-labelled peptide in xenografts of human neuroblastoma. In contrast to 124I-MIBG, which is currently evaluated as a neuroblastoma marker in PET/CT, 68Ga-labelled GD2-specific peptide spared the thyroid but was enriched in the kidneys, which could be partially blocked by infusion of amino acids.In summary, we here report on a novel tumour-homing peptide that specifically binds to the disialoganglioside GD2, accumulates in xenografts of neuroblastoma cells in mice and bears the potential for tumour detection using PET/CT. Thus, this peptide may serve as a new scaffold for diagnosing GD2-positive tumours of neuroectodermal origin. PMID:27716771

  1. How water molecules affect the catalytic activity of hydrolases--a XANES study of the local structures of peptide deformylase.

    PubMed

    Cui, Peixin; Wang, Yu; Chu, Wangsheng; Guo, Xiaoyun; Yang, Feifei; Yu, Meijuan; Zhao, Haifeng; Dong, Yuhui; Xie, Yaning; Gong, Weimin; Wu, Ziyu

    2014-12-12

    Peptide deformylase (PDF) is a prokaryotic enzyme that catalyzes the deformylation of nascent peptides generated during protein synthesis and water molecules play a key role in these hydrolases. Using X-ray absorption near edge spectroscopy (XANES) and ab initio calculations we accurately probe the local atomic environment of the metal ion binding in the active site of PDF at different pH values and with different metal ions. This new approach is an effective way to monitor existing correlations among functions and structural changes. We show for the first time that the enzymatic activity depends on pH values and metal ions via the bond length of the nearest coordinating water (Wat1) to the metal ion. Combining experimental and theoretical data we may claim that PDF exhibits an enhanced enzymatic activity only when the distance of the Wat1 molecule with the metal ion falls in the limited range from 2.15 to 2.55 Å.

  2. Anion transport properties of amine and amide-sidechained peptides are affected by charge and phospholipid composition†

    PubMed Central

    You, Lei; Li, Ruiqiong; Gokel, George W.

    2009-01-01

    Four synthetic anion transporters (SATs) having the general formula (n-C18H37)2N-COCH2OCH2CO-(Gly)3Pro-Lys(ε-N-R)-(Gly)2-O-n-C7H15 were prepared and studied. The group R was Cbz, H (TFA salt), t-Boc, and dansyl in peptides 1, 2, 3, and 4 respectively. The glutamine analog (GGGPQAG sequence) was also included. A dansyl-substituted fluorescent SAT was used to probe peptide insertion; the dansyl sidechain resides in an environment near the bilayer’s midpolar regime. When the lysine sidechain was free or protected amine, little effect was noted on final Cl− transport rate in DOPC : DOPA (7 : 3) liposomes. This stands in contrast to the significant retardation of transport previously observed when a negative glutamate residue was present in the peptide sequence. It was also found that Cl− release from liposomes depended on the phospholipid composition of the vesicles. Chloride transport diminished significantly for the free lysine containing SAT, 2, when the lipid was altered from DOPC : DOPA to pure DOPC. Amide-sidechained SATs 1 and 5 showed a relatively small decrease in Cl− transport. The effect of lipid composition on Cl− transport was explained by differences in electrostatic interaction between amino acid sidechain and lipid headgroup, which was modeled by computation. PMID:18688484

  3. Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections.

    PubMed

    Lefmann, Michael; Schweickert, Birgitta; Buchholz, Petra; Göbel, Ulf B; Ulrichs, Timo; Seiler, Peter; Theegarten, Dirk; Moter, Annette

    2006-10-01

    With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplification-based methods. All isolates (M. tuberculosis complex, n = 24; M. avium, n = 7; M. kansasii, n = 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for species-specific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology.

  4. Identification in pituitary tissue of a peptide alpha-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid.

    PubMed

    Eipper, B A; Mains, R E; Glembotski, C C

    1983-08-01

    An enzymatic activity capable of producing an alpha-amidated peptide product from its glycine-extended precursor has been identified in secretory granules of rat anterior, intermediate, and neural pituitary and bovine intermediate pituitary. High levels of endogenous inhibitors of this alpha-amidation activity have also been found in tissue homogenates. The alpha-amidation activity is totally inhibited by addition of divalent metal ion chelators such as diethyldithiocarbamate, o-phenanthroline, and EDTA; alpha-amidation activity is restored to above control levels upon addition of copper. The alpha-amidation reaction requires the presence of molecular oxygen. Of the various cofactors tested, ascorbic acid was the most potent stimulator of alpha-amidation. The alpha-amidation activity has a neutral pH optimum and is primarily soluble following several cycles of freezing and thawing. Kinetic studies with the bovine intermediate pituitary granule-associated activity demonstrated a linear Lineweaver-Burk plot when D-Tyr-Val-Gly was the varied substrate; the apparent Km and Vmax varied with the concentration of ascorbic acid. The substrate specificity of the alpha-amidation activity appears to be quite broad; the conversion of D-Tyr-Val-Gly into D-Tyr-Val-NH2 is inhibited by the addition of a variety of glycine-extended peptides.

  5. The identification and structure-activity relations of a cardioactive FMRFamide-related peptide from the blue crab Callinectes sapidus.

    PubMed

    Krajniak, K G

    1991-01-01

    The pericardial organs and thoracic ganglia of the blue crab Callinectes sapidus were resected and extracted. The extracts were fractionated by HPLC and the fractions analyzed by a radioimmunoassay (RIA) to FMRFamide. Multiple peaks of immunoreactivity were present and one of these, upon fast atom bombardment mass spectrometry (FAB-ms) and microsequencing, yielded the sequence GYNRSFLRFamide. The amount of this peptide in each crab is between 7 and 13 pmol. Several incomplete sequences were also characterized, suggesting a precursor with multiple copies of peptides related to GYNRSFLRFamide might occur. The peptide caused a dose-dependent increase in heart rate; threshold was 10 to 30 nM, and the EC50 was 323 +/- 62 nM. A structure-activity study of GYNRSFLRFamide on the crab heart suggests that, for full potency, a peptide should be at least a heptapeptide with the sequence XXZFLRFamide, where X is any amino acid and Z is either asparagine or serine.

  6. Isolation and identification of a cAMP generating peptide from the flesh fly, Neobellieria bullata (Diptera: Sarcophagidae).

    PubMed

    Spittaels, K; Devreese, B; Schoofs, L; Neven, H; Janssen, I; Grauwels, L; Van Beeumen, J; De Loof, A

    1996-01-01

    The Manduca sexta Malpighian tubule assay system, developed to monitor adenylate cyclase activity, was used in combination with HPLC to isolate a novel cAMP generating peptide from 350,000 whole flesh flies, Neobellieria bullata. Mass spectrometry revealed a molecular mass of 5,047 daltons, and Edman degradation the following sequence: AGAEAEKLSGLSKYFNGTTMAGRANVAKATYAVIGLIIAYNVMKPKKK. This 48-mer peptide, called Neb-cGP, does not belong to the corticotropin releasing factor family of insect diuretic peptides. Electrophoresis and subsequent immunoblotting of peptides immunoprecipitated from a homogenate of entire flies showed that one fly contained approximately 0.003 to 0.03 micrograms Neb-cGP and that 10 micrograms represents the lowest immunostainable amount on a Western blot.

  7. Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors

    PubMed Central

    Mandelin, Jami; Cardó-Vila, Marina; Driessen, Wouter H. P.; Mathew, Paul; Navone, Nora M.; Lin, Sue-Hwa; Logothetis, Christopher J.; Rietz, Anna Cecilia; Dobroff, Andrey S.; Proneth, Bettina; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand–receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer. PMID:25762070

  8. Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors.

    PubMed

    Mandelin, Jami; Cardó-Vila, Marina; Driessen, Wouter H P; Mathew, Paul; Navone, Nora M; Lin, Sue-Hwa; Logothetis, Christopher J; Rietz, Anna Cecilia; Dobroff, Andrey S; Proneth, Bettina; Sidman, Richard L; Pasqualini, Renata; Arap, Wadih

    2015-03-24

    We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand-receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer. PMID:25762070

  9. Identification and biochemical characterization of a new antibacterial and antifungal peptide derived from the insect Sphodromantis viridis.

    PubMed

    Zare-Zardini, Hadi; Taheri-Kafrani, Asghar; Ordooei, Mahtab; Ebrahimi, Leila; Tolueinia, Behnaz; Soleimanizadeh, Mojgan

    2015-04-01

    Antimicrobial peptides are members of the immune system that protect the host from infection. In this study, a potent and structurally novel antimicrobial peptide was isolated and characterized from praying mantis Sphodromantis viridis. This 14-amino acid peptide was purified by RP-HPLC. Tandem mass spectrometry was used for sequencing this peptide, and the results showed that the peptide belongs to the Mastoparan family. The peptide was named Mastoparan-S. Mastoparan-S demonstrated that it has antimicrobial activities against a broad spectrum of microorganisms (Gram-positive and Gram-negative bacteria and fungi), and it was found to be more potent than common antibiotics such as kanamycin. Mastoparan-S showed higher antimicrobial activity against Gram-negative bacteria compared to Gram-positive ones and fungi. The minimum inhibitory concentration (MIC) values of Mastoparan-S are 15.1-28.3 µg/ml for bacterial and 19.3-24.6 µg/ml for fungal pathogens. In addition, this newly described peptide showed low hemolytic activity against human red blood cells. The in vitro cytotoxicity of Mastoparan-S was also evaluated on monolayer of normal human cells (HeLa) by MTT assay, and the results illustrated that Mastoparan-S had significant cytotoxicity at concentrations higher than 40 µg/ml and had no any cytotoxicity at the MIC (≤30 µg/ml). The findings of the present study reveal that this newly described peptide can be introduced as an appropriate candidate for treatment of topical infection.

  10. Identification and biochemical characterization of a new antibacterial and antifungal peptide derived from the insect Sphodromantis viridis.

    PubMed

    Zare-Zardini, Hadi; Taheri-Kafrani, Asghar; Ordooei, Mahtab; Ebrahimi, Leila; Tolueinia, Behnaz; Soleimanizadeh, Mojgan

    2015-04-01

    Antimicrobial peptides are members of the immune system that protect the host from infection. In this study, a potent and structurally novel antimicrobial peptide was isolated and characterized from praying mantis Sphodromantis viridis. This 14-amino acid peptide was purified by RP-HPLC. Tandem mass spectrometry was used for sequencing this peptide, and the results showed that the peptide belongs to the Mastoparan family. The peptide was named Mastoparan-S. Mastoparan-S demonstrated that it has antimicrobial activities against a broad spectrum of microorganisms (Gram-positive and Gram-negative bacteria and fungi), and it was found to be more potent than common antibiotics such as kanamycin. Mastoparan-S showed higher antimicrobial activity against Gram-negative bacteria compared to Gram-positive ones and fungi. The minimum inhibitory concentration (MIC) values of Mastoparan-S are 15.1-28.3 µg/ml for bacterial and 19.3-24.6 µg/ml for fungal pathogens. In addition, this newly described peptide showed low hemolytic activity against human red blood cells. The in vitro cytotoxicity of Mastoparan-S was also evaluated on monolayer of normal human cells (HeLa) by MTT assay, and the results illustrated that Mastoparan-S had significant cytotoxicity at concentrations higher than 40 µg/ml and had no any cytotoxicity at the MIC (≤30 µg/ml). The findings of the present study reveal that this newly described peptide can be introduced as an appropriate candidate for treatment of topical infection. PMID:25869360

  11. Identification and localization of neurohypophysial peptides in the brain of a caecilian amphibian, Typhlonectes natans (Amphibia: Gymnophiona).

    PubMed

    Hilscher-Conklin, C; Conlon, J M; Boyd, S K

    1998-05-01

    The amphibian order Gymnophiona contains more than 150 different species of caecilians. The characterization and distribution of neurohypophysial peptides, however, has not been described for any member of this order. By using high-performance liquid chromatography, radioimmunoassay, and mass spectrometry, we identified the peptide arginine vasotocin (AVT) in brain and pituitary extracts from the caecilian Typhlonectes natans. By using immunocytochemistry, we found five populations of AVT-immunoreactive (AVT-ir) cells in the brain of T. natans. AVT-ir cell bodies were located in the preoptic area, amygdala pars medialis, ventral thalamus, dorsal hypothalamic nucleus, and nucleus of the solitary tract. AVT-ir fibers and terminal fields were widespread. We also identified a mesotocin-like peptide. The distribution of this peptide in the brain of T. natans was more restricted than the distribution of AVT. Mesotocin-like-immunoreactive cell bodies were located almost exclusively in the preoptic area, with only a few other cells located in the amygdala pars medialis. This caecilian species, therefore, possesses neurohypophysial peptides that are similar in their structure and distribution to the peptides found in anuran and urodele amphibian orders.

  12. Identification of Fusarium virguliforme FvTox1-Interacting Synthetic Peptides for Enhancing Foliar Sudden Death Syndrome Resistance in Soybean.

    PubMed

    Wang, Bing; Swaminathan, Sivakumar; Bhattacharyya, Madan K

    2015-01-01

    Soybean is one of the most important crops grown across the globe. In the United States, approximately 15% of the soybean yield is suppressed due to various pathogen and pests attack. Sudden death syndrome (SDS) is an emerging fungal disease caused by Fusarium virguliforme. Although growing SDS resistant soybean cultivars has been the main method of controlling this disease, SDS resistance is partial and controlled by a large number of quantitative trait loci (QTL). A proteinacious toxin, FvTox1, produced by the pathogen, causes foliar SDS. Earlier, we demonstrated that expression of an anti-FvTox1 single chain variable fragment antibody resulted in reduced foliar SDS development in transgenic soybean plants. Here, we investigated if synthetic FvTox1-interacting peptides, displayed on M13 phage particles, can be identified for enhancing foliar SDS resistance in soybean. We screened three phage-display peptide libraries and discovered four classes of M13 phage clones displaying FvTox1-interacting peptides. In vitro pull-down assays and in vivo interaction assays in yeast were conducted to confirm the interaction of FvTox1 with these four synthetic peptides and their fusion-combinations. One of these peptides was able to partially neutralize the toxic effect of FvTox1 in vitro. Possible application of the synthetic peptides in engineering SDS resistance soybean cultivars is discussed. PMID:26709700

  13. Identification and localization of neurohypophysial peptides in the brain of a caecilian amphibian, Typhlonectes natans (Amphibia: Gymnophiona).

    PubMed

    Hilscher-Conklin, C; Conlon, J M; Boyd, S K

    1998-05-01

    The amphibian order Gymnophiona contains more than 150 different species of caecilians. The characterization and distribution of neurohypophysial peptides, however, has not been described for any member of this order. By using high-performance liquid chromatography, radioimmunoassay, and mass spectrometry, we identified the peptide arginine vasotocin (AVT) in brain and pituitary extracts from the caecilian Typhlonectes natans. By using immunocytochemistry, we found five populations of AVT-immunoreactive (AVT-ir) cells in the brain of T. natans. AVT-ir cell bodies were located in the preoptic area, amygdala pars medialis, ventral thalamus, dorsal hypothalamic nucleus, and nucleus of the solitary tract. AVT-ir fibers and terminal fields were widespread. We also identified a mesotocin-like peptide. The distribution of this peptide in the brain of T. natans was more restricted than the distribution of AVT. Mesotocin-like-immunoreactive cell bodies were located almost exclusively in the preoptic area, with only a few other cells located in the amygdala pars medialis. This caecilian species, therefore, possesses neurohypophysial peptides that are similar in their structure and distribution to the peptides found in anuran and urodele amphibian orders. PMID:9552122

  14. Use of the "blue halo" assay in the identification of genes encoding exported proteins with cleavable signal peptides: cloning of a Borrelia burgdorferi plasmid gene with a signal peptide.

    PubMed Central

    Giladi, M; Champion, C I; Haake, D A; Blanco, D R; Miller, J F; Miller, J N; Lovett, M A

    1993-01-01

    genes encoding proteins with cleavable signal peptides and therefore can serve as a first step in the identification of genes encoding potential virulence factors. Images PMID:8320228

  15. ProPepper: a curated database for identification and analysis of peptide and immune-responsive epitope composition of cereal grain protein families

    PubMed Central

    Juhász, Angéla; Haraszi, Réka; Maulis, Csaba

    2015-01-01

    ProPepper is a database that contains prolamin proteins identified from true grasses (Poaceae), their peptides obtained with single- and multi-enzyme in silico digestions as well as linear T- and B-cell-specific epitopes that are responsible for wheat-related food disorders. The integrated database and analysis platform contains datasets that are collected from multiple public databases (UniprotKB, IEDB, NCBI GenBank), manually curated and annotated, and interpreted in three main data tables: Protein-, Peptide- and Epitope list views that are cross-connected by unique identifications. Altogether 21 genera and 80 different species are represented. Currently, the database contains 2146 unique and complete protein sequences related to 2618 GenBank entries and 35 657 unique peptide sequences that are a result of 575 110 unique digestion events obtained by in silico digestion methods involving six proteolytic enzymes and their combinations. The interface allows advanced global and parametric search functions along with a download option, with direct connections to the relevant public databases. Database URL: https://propepper.net PMID:26450949

  16. Moving Away from the Reference Genome: Evaluating a Peptide Sequencing Tagging Approach for Single Amino Acid Polymorphism Identifications in the Genus Populus

    SciTech Connect

    Abraham, Paul E; Adams, Rachel M; Tuskan, Gerald A; Hettich, Robert {Bob} L

    2013-01-01

    The genetic diversity across natural populations of the model organism, Populus, is extensive, containing a single nucleotide polymorphism roughly every 200 base pairs. When deviations from the reference genome occur in coding regions, they can impact protein sequences. Rather than relying on a static reference database to profile protein expression, we employed a peptide sequence tagging (PST) approach capable of decoding the plasticity of the Populus proteome. Using shotgun proteomics data from two genotypes of P. trichocarpa, a tag-based approach enabled the detection of 6,653 unexpected sequence variants. Through manual validation, our study investigated how the most abundant chemical modification (methionine oxidation) could masquerade as a sequence variant (AlaSer) when few site-determining ions existed. In fact, precise localization of an oxidation site for peptides with more than one potential placement was indeterminate for 70% of the MS/MS spectra. We demonstrate that additional fragment ions made available by high energy collisional dissociation enhances the robustness of the peptide sequence tagging approach (81% of oxidation events could be exclusively localized to a methionine). We are confident that augmenting fragmentation processes for a PST approach will further improve the identification of single amino acid polymorphism in Populus and potentially other species as well.

  17. ProPepper: a curated database for identification and analysis of peptide and immune-responsive epitope composition of cereal grain protein families.

    PubMed

    Juhász, Angéla; Haraszi, Réka; Maulis, Csaba

    2015-01-01

    ProPepper is a database that contains prolamin proteins identified from true grasses (Poaceae), their peptides obtained with single- and multi-enzyme in silico digestions as well as linear T- and B-cell-specific epitopes that are responsible for wheat-related food disorders. The integrated database and analysis platform contains datasets that are collected from multiple public databases (UniprotKB, IEDB, NCBI GenBank), manually curated and annotated, and interpreted in three main data tables: Protein-, Peptide- and Epitope list views that are cross-connected by unique identifications. Altogether 21 genera and 80 different species are represented. Currently, the database contains 2146 unique and complete protein sequences related to 2618 GenBank entries and 35 657 unique peptide sequences that are a result of 575 110 unique digestion events obtained by in silico digestion methods involving six proteolytic enzymes and their combinations. The interface allows advanced global and parametric search functions along with a download option, with direct connections to the relevant public databases. Database URL: https://propepper.net.

  18. Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus.

    PubMed

    Gottschalk, Sanne; Gottlieb, Caroline T; Vestergaard, Martin; Hansen, Paul R; Gram, Lone; Ingmer, Hanne; Thomsen, Line E

    2015-12-01

    The rapid rise in antibiotic-resistant pathogens is causing increased health concerns, and consequently there is an urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs), which have been isolated from a wide range of organisms, represent a very promising class of novel antimicrobials. In the present study, the analogue FL9, based on the amphibian AMP fallaxin, was studied to elucidate its mode of action and antibacterial activity against the human pathogen Staphylococcus aureus. Our data showed that FL9 may have a dual mode of action against S. aureus. At concentrations around the MIC, FL9 bound DNA, inhibited DNA synthesis and induced the SOS DNA damage response, whereas at concentrations above the MIC the interaction between S. aureus and FL9 led to membrane disruption. The antibacterial activity of the peptide was maintained over a wide range of NaCl and MgCl(2) concentrations and at alkaline pH, while it was compromised by acidic pH and exposure to serum. Furthermore, at subinhibitory concentrations of FL9, S. aureus responded by increasing the expression of two major virulence factor genes, namely the regulatory rnaIII and hla, encoding α-haemolysin. In addition, the S. aureus-encoded natural tolerance mechanisms included peptide cleavage and the addition of positive charge to the cell surface, both of which minimized the antimicrobial activity of FL9. Our results add new information about FL9 and its effect on S. aureus, which may aid in the future development of analogues with improved therapeutic potential.

  19. Identification of formyl peptides from Listeria monocytogenes and Staphylococcus aureus as potent chemoattractants for mouse neutrophils 1

    PubMed Central

    Southgate, Erica L.; He, Rong L.; Gao, Ji-Liang; Murphy, Philip M.; Nanamori, Masakatsu; Ye, Richard D.

    2009-01-01

    The prototypic formyl peptide N-formyl-Met-Leu-Phe (fMLF) is a major chemoattractant found in Escherichia coli culture supernatants and a potent agonist at human formyl peptide receptor (FPR) 1. Consistent with this, fMLF induces bactericidal functions in human neutrophils at nanomolar concentrations. However, it is a much less potent agonist for mouse FPR (mFPR) 1 and mouse neutrophils, requiring micromolar concentrations for cell activation. To determine whether other bacteria produce more potent agonists for mFPR1, we examined formyl peptides from Listeria monocytogenes and Staphylococcus aureus for their abilities to activate mouse neutrophils. A pentapeptide (N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL)) from L. monocytogenes and a tetrapeptide (N-formyl-Met-Ile-Phe-Leu (fMIFL)) from S. aureus were found to induce mouse neutrophil chemotaxis at 1-10 nM and superoxide production at 10-100 nM, similar to the potency of fMLF on human neutrophils. Using transfected cell lines expressing mFPR1 and mFPR2, which are major forms of FPRs in mouse neutrophils, we found that mFPR1 is responsible for the high potency of fMIVIL and fMIFL. In comparison, activation of mFPR2 requires micromolar concentrations of the two peptides. Genetic deletion of mfpr1 resulted in abrogation of neutrophil superoxide production and degranulation in response to fMIVIL and fMIFL, further demonstrating that mFPR1 is the primary receptor for detection of these formyl peptides. In conclusion, the formyl peptides from L. monocytogenes and S. aureus are 100-fold more potent than fMLF in activating mouse neutrophils. The ability of mFPR1 to detect bacterially derived formyl peptides indicates that this important host defense mechanism is conserved in mice. PMID:18606697

  20. Does Thermal Breathing Affect Collision Cross Sections of Gas-Phase Peptide Ions? An Ab Initio Molecular Dynamics Study.

    PubMed

    Pepin, Robert; Petrone, Alessio; Laszlo, Kenneth J; Bush, Matthew F; Li, Xiaosong; Tureček, František

    2016-07-21

    Ab initio molecular dynamics (AIMD) with density functional theory (DFT) was applied to explore conformational motions and collision cross sections (Ω) of folded (2) and extended (7) conformers of doubly charged peptide ions, (Ala-Ala-Leu-Arg + 2H)(2+), in the gas phase at 300 and 473 K. The experimental Ω of (Ala-Ala-Leu-Arg +2H)(2+) was measured as 149 ± 1.2 Å(2) at 298 K. Thermally distributed mean values of Ω for 2 and 7 at 300 and 473 K were only 0.8-1.1% larger than for the equilibrium 0 K structures. Long (>10 ps) trajectory calculations indicated entropy-driven conformational change of 2 to 7 that occurred at random within a ∼ 4 ps time window. The experimental Ω was found to fit the calculated population averaged values for 2 and 7, indicating a rapid conformer interconversion. Overall, thermal breathing had only a minor effect on the peptide ion collision cross sections.

  1. Pipa carvalhoi skin secretion profiling: absence of peptides and identification of kynurenic acid as the major constitutive component.

    PubMed

    Mariano, Douglas Oscar Ceolin; Yamaguchi, Lydia Fumiko; Jared, Carlos; Antoniazzi, Marta Maria; Sciani, Juliana Mozer; Kato, Massuo Jorge; Pimenta, Daniel Carvalho

    2015-01-01

    The presence of peptides has been identified in all African pipid genera; nevertheless, little is known about skin secretion of South American frog genus Pipa. Skin secretion from captive and wild Pipa carvalhoi were obtained in the presence or absence of norepinephrine stimulation. The <10 kDa fraction was analyzed by liquid chromatography and mass spectrometry, searching for peptides. Chromatographic profiles show the presence of a major component in this secretion, regardless of the stimulation method (norepinephrine or mechanical stimulation) and the origin of the animal (captivity or wild), as well as in the absence of any stimulus. The general mass distribution profile in P. carvalhoi skin secretion shows numerous components below 800 Da. Moreover, no peptide could be identified, regardless of the chromatographic approach. The major component was purified and identified as kynurenic acid, an L-tryptophan derivative. P. carvalhoi does not secrete peptides as toxins in its skin. In addition, we here report that kynurenic acid is the main component of P. carvalhoi skin secretion. Although no biological activity was associated with kynurenic acid, we propose that this molecule is a pheromone that signals the presence of a co-specific in the shady environment in which this animal lives. In this study we demonstrate the absence of peptidic toxins in the skin secretion of P. carvalhoi, a break of paradigm in the pipid family.

  2. PEP-SiteFinder: a tool for the blind identification of peptide binding sites on protein surfaces.

    PubMed

    Saladin, Adrien; Rey, Julien; Thévenet, Pierre; Zacharias, Martin; Moroy, Gautier; Tufféry, Pierre

    2014-07-01

    Peptide-protein interactions are important to many processes of life, particularly for signal transmission or regulatory mechanisms. When no information is known about the interaction between a protein and a peptide, it is of interest to propose candidate sites of interaction at the protein surface, to assist the design of biological experiments to probe the interaction, or to serve as a starting point for more focused in silico approaches. PEP-SiteFinder is a tool that will, given the structure of a protein and the sequence of a peptide, identify protein residues predicted to be at peptide-protein interface. PEP-SiteFinder relies on the 3D de novo generation of peptide conformations given its sequence. These conformations then undergo a fast blind rigid docking on the complete protein surface, and we have found, as the result of a benchmark over 41 complexes, that the best poses overlap to some extent the experimental patch of interaction for close to 90% complexes. In addition, PEP-SiteFinder also returns a propensity index we have found informative about the confidence of the prediction. The PEP-SiteFinder web server is available at http://bioserv.rpbs.univ-paris-diderot.fr/PEP-SiteFinder.

  3. Identification of small-molecule binding pockets in the soluble monomeric form of the Aβ42 peptide

    PubMed Central

    Zhu, Maximillian; Simone, Alfonso De; Schenk, Dale; Toth, Gergely; Dobson, Christopher M.; Vendruscolo, Michele

    2016-01-01

    The aggregation of intrinsically disordered peptides and proteins is associated with a wide range of highly debilitating neurological and systemic disorders. In this work we explored the potential of a structure-based drug discovery procedure to target one such system, the soluble monomeric form of the Aβ42 peptide. We utilised for this purpose a set of structures of the Aβ42 peptide selected from clusters of conformations within an ensemble generated by molecular dynamics simulations. Using these structures we carried out fragment mapping calculations to identify binding ‘hot spots’ on the monomeric form of the Aβ42 peptide. This procedure provided a set of hot spots with ligand efficiencies comparable to those observed for structured proteins, and that are clustered into binding pockets. We verified that such pockets exhibit a propensity to bind small molecules known to interact with the Aβ42 peptide. Taken together these results provide an initial indication that fragment-based drug discovery may represent a potential therapeutic strategy for diseases associated with the aggregation of intrinsically disordered proteins. PMID:23883055

  4. Identification of a novel cathelicidin antimicrobial peptide from ducks and determination of its functional activity and antibacterial mechanism

    PubMed Central

    Gao, Wei; Xing, Liwei; Qu, Pei; Tan, Tingting; Yang, Na; Li, Dan; Chen, Huixian; Feng, Xingjun

    2015-01-01

    The family of antimicrobial peptide, cathelicidins, which plays important roles against infections in animals, has been identified from many species. Here, we identified a novel avian cathelicidin ortholog from ducks and named dCATH. The cDNA sequence of dCATH encodes a predicted 146-amino-acid polypeptide composed of a 17-residue signal peptide, a 109-residue conserved cathelin domain and a 20-residue mature peptide. Phylogenetic analysis demonstrated that dCATH is highly divergent from other avian peptides. The α-helical structure of the peptide exerted strong antimicrobial activity against a broad range of bacteria in vitro, with most minimum inhibitory concentrations in the range of 2 to 4 μM. Moreover, dCATH also showed cytotoxicity, lysing 50% of mammalian erythrocytes in the presence or absence of 10% fetal calf serum at concentrations of 32 μM or 20 μM and killing 50% HaCaT cells at a concentration of 10 μM. The effects on bacterial outer and inner membranes, as examined by scanning electron microscope and transmission electron microscopy, indicate that dCATH kills microbial cells by increasing permeability, causing a loss of membrane integrity. PMID:26608073

  5. Identification of a NEP1-35 recognizing peptide that neutralizes CNS myelin inhibition using phage display library.

    PubMed

    Deng, Qiyue; Cai, Wenqin; Li, Shurong; Su, Bingyin

    2013-03-01

    Nogo-A has been identified as an inhibitory molecule to neurite outgrowth after injury in adult mammalian central nervous system (CNS). The C-terminal fragment of Nogo-A, Nogo-66, inhibits axonal regrowth through NgR1 signaling. Residues 1-32 of Nogo-66 cover two regions that contribute most affinity of Nogo-66 to NgR1. It is unclear whether blocking the two regions with specific small ligands could neutralize the inhibition of Nogo-66. Therefore in this study we explored two phage display peptide libraries to screen small peptides that might bind Nogo-66. NEP1-35 containing 1-33 residues of Nogo-66 was taken as the target for panning. We found that phage-borne peptides with stronger affinity to NEP1-35 contained a relatively conserved motif, RRXXXXXXXRRX. Afterwards one identified peptide, NH(2)-RRQTLSHQMRRP-COOH was synthesized and tested in neurite outgrowth assay, in which this small molecule showed moderate ability to neutralize CNS myelin inhibition in vitro. Our results demonstrated that short peptides could act as adaptors to Nogo-66 and neutralize CNS myelin inhibition in vitro. Additionally, the results also suggested that phage display could help to discover novel small molecules with high affinity to CNS regrowth inhibitors, which might be able to promote CNS regeneration with fewer side effects since they could block only the corresponding regions of inhibitors.

  6. Comprehensive peptide marker identification for the detection of multiple nut allergens using a non-targeted LC-HRMS multi-method.

    PubMed

    Korte, Robin; Lepski, Silke; Brockmeyer, Jens

    2016-05-01

    Food allergies have emerged as a global problem over the last few decades; therefore, reliable and sensitive analytical methods to ensure food safety for allergic consumers are required. The application of mass spectrometry is of growing interest in this field and several procedures based on low resolution tandem mass spectrometry using single tryptic peptides as analytical targets have recently been described. However, a comprehensive survey of marker peptides for the development of multi-methods is still missing, as is a consensus guide to marker identification. In this study, we therefore report a consistent approach to the development of liquid chromatography-mass spectrometry (LC-MS) multi-screening methods for the detection of allergens in food matrices. Proteotypic peptides were identified by a shotgun proteomics approach and verified through a thorough investigation of specificity and sensitivity. On the basis of this procedure, we identified 44 suitable tryptic marker peptides from six allergenic nut species and developed the first analytical LC-MS method for the detection of trace nut contaminations in processed foods using high resolution mass spectrometry (HRMS). The analysis of spiked matrix samples gave limits of detection (LODs) below 10 μg/g for several nuts; these LODs are comparable with routinely used methods such as ELISA and PCR. Notably, the HRMS approach can be used in an untargeted fashion to identify multiple allergens also retrospectively. In conclusion, we present here the so far largest consensus set of analytical markers from nut allergens and to the best of our knowledge the first multi-allergen method based on LC-HRMS.

  7. Identification of vulnerable sites in salts affected agricultural soils from South-Eastern Spain

    NASA Astrophysics Data System (ADS)

    Acosta, Jose A.; Faz, Angel; Kalbitz, Karsten; Jansen, Boris; Silvia, Martinez-Martinez

    2010-05-01

    little adsorption to soil colloidal particles. However, other ions such as sulfate, calcium, magnesium, and sodium also displayed significant increases in concentration in July. This can be explained by the movements of soluble salt to the surface due to evaporation and capillary rise and subsequent precipitation of the salts during high temperatures and low rainfall. Rainfall or irrigation events enhance the leaching of salts to deeper soil horizons. The most affected area is located in the west of the study area, at the lowest altitude within the study area. Depressions favour accumulation of salts, due to both runoffs from higher areas during rainfall periods and poor quality irrigation water. It is recommended to use a better quality of water, at least before the summer, in order to reduce the amount of salts in the surface layer, likely to cause stress to crops growing on the soil in question. In conclusion, the spatial distribution of anions in the soil solution is very useful for predicting where higher increases in salinity will be produced. This will allow for identification of vulnerable areas and subsequent implementation of the necessary measures to decrease the risk for sensitive crops. Acknowledgements: to "Fundación Séneca" of "Comunidad Autónoma de Murcia" for its financial support.

  8. HPLC-Q-TOF-MS identification of antioxidant and antihypertensive peptides recovered from cherry (Prunus cerasus L.) subproducts.

    PubMed

    García, María Concepción; Endermann, Jochan; González-García, Estefanía; Marina, María Luisa

    2015-02-11

    The processing of fruits, such as cherries, is characterized by generating a lot of waste material such as fruit stones, skins, etc. To contribute to environmental sustainability, it is necessary to recover these residues. Cherry stones contain seeds with a significant amount of proteins that are underused and undervalued. The aim of this work was to extract cherry seed proteins, to evaluate the presence of bioactive peptides, and to identify them by mass spectrometry. The digestion of cherry seed proteins was optimized, and three different enzymes were employed: Alcalase, Thermolysin, and Flavourzyme. Peptide extracts obtained by the digestion of the cherry seed protein isolate with Alcalase and Thermolysin yielded the highest antioxidant and antihypertensive capacities. Ultrafiltration of hydrolysates allowed obtaining fractions with high antioxidant and antihypertensive capabilities. HPLC-Q-TOF-MS together with bioinformatics tools enabled one to identify peptides in these fractions.

  9. HPLC-Q-TOF-MS identification of antioxidant and antihypertensive peptides recovered from cherry (Prunus cerasus L.) subproducts.

    PubMed

    García, María Concepción; Endermann, Jochan; González-García, Estefanía; Marina, María Luisa

    2015-02-11

    The processing of fruits, such as cherries, is characterized by generating a lot of waste material such as fruit stones, skins, etc. To contribute to environmental sustainability, it is necessary to recover these residues. Cherry stones contain seeds with a significant amount of proteins that are underused and undervalued. The aim of this work was to extract cherry seed proteins, to evaluate the presence of bioactive peptides, and to identify them by mass spectrometry. The digestion of cherry seed proteins was optimized, and three different enzymes were employed: Alcalase, Thermolysin, and Flavourzyme. Peptide extracts obtained by the digestion of the cherry seed protein isolate with Alcalase and Thermolysin yielded the highest antioxidant and antihypertensive capacities. Ultrafiltration of hydrolysates allowed obtaining fractions with high antioxidant and antihypertensive capabilities. HPLC-Q-TOF-MS together with bioinformatics tools enabled one to identify peptides in these fractions. PMID:25599260

  10. Identification of small peptides inhibiting the integrase-LEDGF/p75 interaction through targeting the cellular co-factor.

    PubMed

    Cavalluzzo, Claudia; Christ, Frauke; Voet, Arnout; Sharma, Ajendra; Singh, Brajendra Kumar; Zhang, Kam Y J; Lescrinier, Eveline; De Maeyer, Marc; Debyser, Zeger; Van der Eycken, Erik

    2013-10-01

    The integration of the viral DNA into the host genome is one of the essential steps in the HIV replication cycle. This process is mediated by the viral enzyme integrase (IN) and lens epithelium-derived growth factor (LEDGF/p75). LEDGF/p75 has been identified as a crucial cellular co-factor of integration that acts by tethering IN to the cellular chromatin. Recently, circular peptides were identified that bind to the C-terminal domain of IN and disrupt the interaction with LEDGF/p75. Starting from the circular peptides, we identified a short peptidic sequence able to inhibit the LEDGF/p75-IN interaction at low μM concentration through its binding to the IN binding site of LEDGF/p75. This discovery can lead to the synthesis of peptidomimetics with high anti-HIV activity targeting the cellular co-factor LEDGF/p75 and not the viral protein IN.

  11. Identification of SYWKQCAFNAVSCFamide: a broadly conserved crustacean C-type allatostatin-like peptide with both neuromodulatory and cardioactive properties

    PubMed Central

    Dickinson, Patsy S.; Wiwatpanit, Teerawat; Gabranski, Emily R.; Ackerman, Rachel J.; Stevens, Jake S.; Cashman, Christopher R.; Stemmler, Elizabeth A.; Christie, Andrew E.

    2009-01-01

    Summary The allatostatins comprise three structurally distinct peptide families that regulate juvenile hormone production by the insect corpora allata. A-type family members contain the C-terminal motif –YXFGLamide and have been found in species from numerous arthropod taxa. Members of the B-type family exhibit a –WX6Wamide C-terminus and, like the A-type peptides, appear to be broadly conserved within the Arthropoda. By contrast, members of the C-type family, typified by the unblocked C-terminus –PISCF, a pyroglutamine blocked N-terminus, and a disulfide bridge between two internal Cys residues, have only been found in holometabolous insects, i.e. lepidopterans and dipterans. Here, using transcriptomics, we have identified SYWKQCAFNAVSCFamide (disulfide bridging predicted between the two Cys residues), a known honeybee and water flea C-type-like peptide, from the American lobster Homarus americanus (infraorder Astacidea). Using matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), a mass corresponding to that of SYWKQCAFNAVSCFamide was detected in the H. americanus brain, supporting the existence of this peptide and its theorized structure. Furthermore, SYWKQCAFNAVSCFamide was detected by MALDI-FTMS in neural tissues from five additional astacideans as well as 19 members of four other decapod infraorders (i.e. Achelata, Anomura, Brachyura and Thalassinidea), suggesting that it is a broadly conserved decapod peptide. In H. americanus, SYWKQCAFNAVSCFamide is capable of modulating the output of both the pyloric circuit of the stomatogastric nervous system and the heart. This is the first demonstration of bioactivity for this peptide in any species. PMID:19423507

  12. Identification and distribution of gonadotropin-releasing hormone-like peptides in the brain of horseshoe crab Tachypleus tridentatus

    NASA Astrophysics Data System (ADS)

    Huang, Huiyang; Li, Linming; Ye, Haihui; Feng, Biyun; Li, Shaojing

    2013-03-01

    Gonadotropin-releasing hormone (GnRH) is a crucial peptide for the regulation of reproduction. Using immunological techniques, we investigated the presence of GnRH in horseshoe crab Tachypleus tridentatus. Octopus GnRH-like immunoreactivity, tunicate GnRH-like immunoreactivity, and lamprey GnRH-I-like immunoreactivity were detected in the neurons and fibers of the protocerebrum. However, no mammal GnRH-like immunoreactivity or lamprey GnRH-III-like immunoreactivity was observed. Our results suggest that a GnRH-like factor, an ancient peptide, existed in the brain of T. tridentatus and may be involved in the reproductive endocrine system.

  13. Identification of genes affecting expression of phosphoglycerate kinase on the surface of group B streptococcus.

    PubMed

    Boone, Tyler J; Tyrrell, Gregory J

    2012-04-01

    Group B streptococcal phosphoglycerate kinase (GBS-PGK), a glycolytic enzyme, has previously been identified on the surface of group B streptococcus (GBS). To identify genes involved in surface expression of GBS-PGK, we performed Tn917 mutagenesis followed by quantification of PGK expressed on the GBS surface. Tn917 mutagenesis identified 4 genes (sag0966, sag0979, sag0980, and sag1003) that when disrupted, alter expression of GBS-PGK on the bacterial surface. Three of the identified genes were localized to a region of the GBS genome containing genes (sag0973-sag0977) predicted to be involved in resistance to antimicrobial peptides. One mutant isolate, designated NCS13sag1003::Tn917, was found to have increased sensitivity to the antimicrobial peptides bacitracin and nisin. In addition, all of the mutant strains assayed were found to have decreased β-hemolysis. In conclusion, we have identified genes involved in surface expression of GBS-PGK. These genes also appear to be involved in antimicrobial peptide resistance and regulate expression of the β-hemolysin. PMID:22444251

  14. Identification of Differentially Expressed Genes through Integrated Study of Alzheimer’s Disease Affected Brain Regions

    PubMed Central

    Berretta, Regina; Moscato, Pablo

    2016-01-01

    Background Alzheimer’s disease (AD) is the most common form of dementia in older adults that damages the brain and results in impaired memory, thinking and behaviour. The identification of differentially expressed genes and related pathways among affected brain regions can provide more information on the mechanisms of AD. In the past decade, several studies have reported many genes that are associated with AD. This wealth of information has become difficult to follow and interpret as most of the results are conflicting. In that case, it is worth doing an integrated study of multiple datasets that helps to increase the total number of samples and the statistical power in detecting biomarkers. In this study, we present an integrated analysis of five different brain region datasets and introduce new genes that warrant further investigation. Methods The aim of our study is to apply a novel combinatorial optimisation based meta-analysis approach to identify differentially expressed genes that are associated to AD across brain regions. In this study, microarray gene expression data from 161 samples (74 non-demented controls, 87 AD) from the Entorhinal Cortex (EC), Hippocampus (HIP), Middle temporal gyrus (MTG), Posterior cingulate cortex (PC), Superior frontal gyrus (SFG) and visual cortex (VCX) brain regions were integrated and analysed using our method. The results are then compared to two popular meta-analysis methods, RankProd and GeneMeta, and to what can be obtained by analysing the individual datasets. Results We find genes related with AD that are consistent with existing studies, and new candidate genes not previously related with AD. Our study confirms the up-regualtion of INFAR2 and PTMA along with the down regulation of GPHN, RAB2A, PSMD14 and FGF. Novel genes PSMB2, WNK1, RPL15, SEMA4C, RWDD2A and LARGE are found to be differentially expressed across all brain regions. Further investigation on these genes may provide new insights into the development of AD

  15. Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's A{beta} peptide

    SciTech Connect

    Garvey, Megan; Tepper, Katharina; Haupt, Caroline; Knuepfer, Uwe; Klement, Karolin; Meinhardt, Jessica; Horn, Uwe; Balbach, Jochen; Faendrich, Marcus

    2011-06-10

    Highlights: {yields} Sodium phosphate buffer accelerated A{beta}(1-40) nucleation relative to HEPES. {yields} A{beta}(1-40) fibrils formed in the two buffers show only minor structural differences. {yields} NMR revealed that A{beta}(1-40) histidine residues mediate buffer dependent changes. -- Abstract: The oligomerization of A{beta} peptide into amyloid fibrils is a hallmark of Alzheimer's disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of A{beta} and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of A{beta} peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of A{beta} fibrillation. The three histidine residues at positions 6, 13 and 14 of A{beta}(1-40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.

  16. Hydrophobic Peptides Affect Binding of Calmodulin and Ca2+ as Explored by H/D Amide Exchange and Mass Spectrometry

    PubMed Central

    Sperry, Justin B.; Huang, Richard Y-C.; Zhu, Mei M.; Rempel, Don L.; Gross, Michael L.

    2010-01-01

    Calmodulin (CaM), a ubiquitous intracellular sensor protein, binds Ca2+ and interacts with various targets as part of signal transduction. Using hydrogen/deuterium exchange (H/DX) and a high resolution PLIMSTEX (Protein-Ligand Interactions by Mass Spectrometry, Titration, and H/D Exchange) protocol, we examined five different states of calmodulin: calcium-free, calcium-loaded, and three states of calcium-loaded in the presence of either melittin, mastoparan, or skeletal myosin light-chain kinase (MLCK). When CaM binds Ca2+, the extent of HDX decreased, consistent with the protein becoming stabilized upon binding. Furthermore, Ca2+-saturated calmodulin exhibits increased protection when bound to the peptides, forming high affinity complexes. The protocol reveals significant changes in EF hands 1, 3, and 4 with saturating levels of Ca2+. Titration of the protein using PLIMSTEX provides the binding affinity of Ca2+ to calmodulin within previously reported values. The affinities of calmodulin to Ca2+ increase by factors of 300 and 1000 in the presence of melittin and mastoparan, respectively. A modified PLIMSTEX protocol whereby the protein is digested to component peptides gives a region-specific titration. The titration data taken in this way show a decrease in the root mean square fit of the residuals, indicating a better fit of the data. The global H/D exchange results and those obtained in a region-specific way provide new insight into the Ca2+-binding properties of this well-studied protein. PMID:21765646

  17. Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes.

    PubMed

    Zhang, Zhenbin; Yan, Xiaojing; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J

    2015-04-21

    A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.

  18. A detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes

    PubMed Central

    Zhang, Zhenbin; Yan, Xiaojing; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J.

    2015-01-01

    A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with on-line pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction, and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3,381 peptides were identified from 50 ng of the digest in a 2.5 hour analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the on-line pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary. PMID:25822566

  19. Identification of a novel peptide ligand targeting visceral adipose tissue via transdermal route by in vivo phage display.

    PubMed

    Lee, Nam Kyung; Kim, Hong Shin; Kim, Kyung Hyun; Kim, Eun-Bae; Cho, Chong Su; Kang, Sang Kee; Choi, Yun Jaie

    2011-11-01

    To find novel peptide ligands targeting visceral adipose tissue (visceral fat) via transdermal route, in vivo phage display screening was conducted by dermal administration of a phage-peptide library to rats and a peptide sequence, CGLHPAFQC (designated as TDA1), was identified as a targeting ligand to visceral adipose tissue through the consecutive transdermal biopannings. Adipocyte-specific affinity and transdermal activity of the TDA1 were validated in vitro and targeting ability of the dermally administered TDA1 to visceral adipose tissue was also confirmed in vivo. TDA1 was effectively translocated into systemic circulation after dermal administration and selectively targeted visceral adipose tissue without any preference to other organs tested. Fluorescent microscopic analysis revealed that the TDA1 could be specifically localized in the hair follicles of the skin, as well as in the visceral adipose tissue. Thus, we inferred that dermally administered TDA1 would first access systemic circulation via hair follicles as its transdermal route and then could target visceral fat effectively. The overall results suggest that the TDA1 peptide could be potentially applied as a homing moiety for delivery of anti-obesity therapeutics to visceral fat through the convenient transdermal pathway. PMID:21999821

  20. MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins

    PubMed Central

    2016-01-01

    Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods. PMID:26985971

  1. Identification of antihyperuricemic peptides in the proteolytic digest of shark cartilage water extract using in vivo activity-guided fractionation.

    PubMed

    Murota, Itsuki; Taguchi, Satoko; Sato, Nobuyuki; Park, Eun Young; Nakamura, Yasushi; Sato, Kenji

    2014-03-19

    A peptide that exerts antihyperuricemic activity after oral administration was identified from a microbial protease (alcalase) digest of the water extract of shark cartilage by in vivo activity-guided fractionation, using oxonate-induced hyperuricemic rats. Water extract of shark cartilage was first fractionated by preparative ampholine-free isoelectric focusing, followed by preparative reversed-phase liquid chromatography. The antihyperuricemic activity of the alcalse digests of the obtained fractions was evaluated using an animal model. Alcalase digests of the basic and hydrophobic fractions exerted antihyperuricemic activity. A total of 18 peptides were identified in the alcalase digest of the final active fraction. These peptides were chemically synthesized and evaluated for antihyperuricemic activity. Tyr-Leu-Asp-Asn-Tyr and Ser-Pro-Pro-Tyr-Trp-Pro-Tyr lowered the serum uric acid level via intravenous injection at 5 mg/kg of body weight. Furthermore, orally administered Tyr-Leu-Asp-Asn-Tyr showed antihyperuricemic activity. Therefore, these peptides are at least partially responsible for the antihyperuricemic activity of the alcalase digest of shark cartilage.

  2. High-throughput fluorescence screening assay for the identification and comparison of antimicrobial peptides' activity on various yeast species.

    PubMed

    Kodedová, Marie; Sychrová, Hana

    2016-09-10

    New antifungal compounds that circumvent the resistance of the pathogen by directly damaging yeast cell surface structures are promising agents for the treatment of fungal infections, due to their different mechanism of action from current clinically used antifungal drugs. We present here a rapid and cost-effective fluorescence method suitable for identifying new potent drugs that directly target yeast cell surface structures, causing cell permeabilization and thus bypassing the multidrug resistance mechanisms of pathogens. The fluorescence assay enabled us to detect with high sensitivity damage to the Candida plasma membrane (its hyperpolarization and permeabilization) as a result of short-term exposure to the antifungal compounds. Results can be obtained in 1-2h with minimal effort and consumption of the tested compounds, also 96 samples can be analysed simultaneously. We used this method to study antimicrobial peptides isolated from the venom of bees and their synthetic analogs, compare the potency of the peptides and determine their minimal effective concentrations. The antimicrobial peptides were able to kill yeast cells at low concentrations within a 15-min treatment, the LL-III peptide exhibited a broad spectrum of antifungal activity on various Saccharomyces, pathogenic Candida and osmotolerant yeast species. PMID:27369550

  3. Identification of a novel peptide ligand targeting visceral adipose tissue via transdermal route by in vivo phage display.

    PubMed

    Lee, Nam Kyung; Kim, Hong Shin; Kim, Kyung Hyun; Kim, Eun-Bae; Cho, Chong Su; Kang, Sang Kee; Choi, Yun Jaie

    2011-11-01

    To find novel peptide ligands targeting visceral adipose tissue (visceral fat) via transdermal route, in vivo phage display screening was conducted by dermal administration of a phage-peptide library to rats and a peptide sequence, CGLHPAFQC (designated as TDA1), was identified as a targeting ligand to visceral adipose tissue through the consecutive transdermal biopannings. Adipocyte-specific affinity and transdermal activity of the TDA1 were validated in vitro and targeting ability of the dermally administered TDA1 to visceral adipose tissue was also confirmed in vivo. TDA1 was effectively translocated into systemic circulation after dermal administration and selectively targeted visceral adipose tissue without any preference to other organs tested. Fluorescent microscopic analysis revealed that the TDA1 could be specifically localized in the hair follicles of the skin, as well as in the visceral adipose tissue. Thus, we inferred that dermally administered TDA1 would first access systemic circulation via hair follicles as its transdermal route and then could target visceral fat effectively. The overall results suggest that the TDA1 peptide could be potentially applied as a homing moiety for delivery of anti-obesity therapeutics to visceral fat through the convenient transdermal pathway.

  4. Identification of the thiazolyl peptide GE37468 gene cluster from Streptomyces ATCC 55365 and heterologous expression in Streptomyces lividans.

    PubMed

    Young, Travis S; Walsh, Christopher T

    2011-08-01

    Thiazolyl peptides are bacterial secondary metabolites that potently inhibit protein synthesis in Gram-positive bacteria and malarial parasites. Recently, our laboratory and others reported that this class of trithiazolyl pyridine-containing natural products is derived from ribosomally synthesized preproteins that undergo a cascade of posttranslational modifications to produce architecturally complex macrocyclic scaffolds. Here, we report the gene cluster responsible for production of the elongation factor Tu (EF-Tu)-targeting 29-member thiazolyl peptide GE37468 from Streptomyces ATCC 55365 and its heterologous expression in the model host Streptomyces lividans. GE37468 harbors an unusual β-methyl-δ-hydroxy-proline residue that may increase conformational rigidity of the macrocycle and impart reduced entropic costs of target binding. Isotope feeding and gene knockout were employed in the engineered S. lividans strain to identify the P450 monooxygenase GetJ as the enzyme involved in posttranslational transformation of isoleucine 8 to β-methyl-δ-hydroxy-proline through a predicted tandem double hydroxylation/cyclization mechanism. Loss of Ile8 oxygenative cyclization or mutation of Ile8 to alanine via preprotein gene replacement resulted in a 4-fold and 2-fold drop in antibiotic activity, respectively. This report of genetic manipulation of a 29-member thiazolyl peptide sets the stage for further genetic examination of structure activity relationships in the EF-Tu targeting class of thiazolyl peptides.

  5. Differential effect of amelogenin peptides on osteogenic differentiation in vitro: identification of possible new drugs for bone repair and regeneration.

    PubMed

    Amin, Harsh D; Olsen, Irwin; Knowles, Jonathan C; Donos, Nikolaos

    2012-06-01

    Enamel matrix proteins (EMP) have been shown to promote regeneration of periodontal ligament and root cementum, and sometimes to enhance the differentiation of bone-forming cells in vitro and new bone growth in vivo. However, the inconsistent and unpredictable effects of EMP that have been reported for bone regeneration may be due to the highly variable composition of this heterogeneous material, which is comprised mainly of amelogenin and amelogenin-derived peptides. The present study has therefore examined the effects of naturally occurring low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions of Emdogain(®) (EMD; Institut Straumann, Basel, Switzerland), a commercially available form of EMP, on osteogenic differentiation of bone precursor cells in vitro. In addition, the effects of chemically synthesized specific components of LMW and HMW-namely, the tyrosine-rich amelogenin peptide (TRAP), a specific amelogenin isoform derived by proteolytic clipping, and a leucine-rich amelogenin peptide (LRAP), an isoform derived by alternative splicing-on bone-forming cell activity were also investigated. Our findings demonstrate that while TRAP suppressed the formation of bone-like mineralized nodules, LRAP upregulated osteogenic differentiation. Furthermore, synthetically produced TRAP and its unique C-terminal 12 amino acid sequence (TCT) also suppressed bone-forming cells, whereas LRAP and its unique C-terminal 23 amino acid sequence (LCT) markedly enhanced terminal differentiation of bone-forming cells. These findings suggest that the differential effects of amelogenin-derived peptide sequences present in EMP could be of potential clinical value, with the novel bioactive TCT peptide as a useful tool for limiting pathological bone cell growth and the unique LCT sequence having therapeutic benefits in the treatment of periodontal and orthopedic diseases. PMID:22320389

  6. Identification of new leishmanicidal peptide lead structures by automated real-time monitoring of changes in intracellular ATP.

    PubMed Central

    Luque-Ortega, J Román; Saugar, José M; Chiva, Cristina; Andreu, David; Rivas, Luis

    2003-01-01

    Leishmanicidal drugs interacting stoichiometrically with parasite plasma membrane lipids, thus promoting permeability, have raised significant expectations for Leishmania chemotherapy due to their nil or very low induction of resistance. Inherent in this process is a decrease in intracellular ATP, either wasted by ionic pumps to restore membrane potential or directly leaked through larger membrane lesions caused by the drug. We have adapted a luminescence method for fast automated real-time monitoring of this process, using Leishmania donovani promastigotes transfected with a cytoplasmic luciferase form, previously tested for anti-mitochondrial drugs. The system was first assayed against a set of well-known membrane-active drugs [amphotericin B, nystatin, cecropin A-melittin peptide CA(1-8)M(1-18)], plus two ionophoric polyethers (narasin and salinomycin) not previously tested on Leishmania, then used to screen seven new cecropin A-melittin hybrid peptides. All membrane-active compounds showed a good correlation between inhibition of luminescence and leishmanicidal activity. Induction of membrane permeability was demonstrated by dissipation of membrane potential, SYTOX trade mark Green influx and membrane damage assessed by electron microscopy, except for the polyethers, where ATP decrease was due to inhibition of its mitochondrial synthesis. Five of the test peptides showed an ED50 around 1 microM on promastigotes. These peptides, with equal or better activity than 26-residue-long CA(1-8)M(1-18), are the shortest leishmanicidal peptides described so far, and validate our luminescence assay as a fast and cheap screening tool for membrane-active compounds. PMID:12864731

  7. Identification of the sAPRIL Binding Peptide and Its Growth Inhibition Effects in the Colorectal Cancer Cells

    PubMed Central

    Liu, Fang; Li, Jing; He, Mei-rong

    2015-01-01

    Background A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) super family. It binds to its specific receptors and is involved in multiple processes during tumorigenesis and tumor cells proliferation. High levels of APRIL expression are closely correlated to the growth, metastasis, and 5-FU drug resistance of colorectal cancer. The aim of this study was to identify a specific APRIL binding peptide (BP) able to block APRIL activity that could be used as a potential treatment for colorectal cancer. Methods A phage display library was used to identify peptides that bound selectively to soluble recombinant human APRIL (sAPRIL). The peptides with the highest binding affinity for sAPRIL were identified using ELISA. The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively. An in vivo mouse model of colorectal cancer was used to determine the anti-tumor efficacy of the sAPRIL-BP. Results Three candidate peptides were characterized from eight phage clones with high binding affinity for sAPRIL. The peptide with the highest affinity was selected for further characterization. The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner. In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally. Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells. Conclusions sAPRIL-BP significantly suppressed tumor growth in vitro and in vivo and might be a candidate for treating colorectal cancers that express high levels of APRIL. PMID:25826583

  8. Computational peptide vaccinology.

    PubMed

    Söllner, Johannes

    2015-01-01

    Immunoinformatics focuses on modeling immune responses for better understanding of the immune system and in many cases for proposing agents able to modify the immune system. The most classical of these agents are vaccines derived from living organisms such as smallpox or polio. More modern vaccines comprise recombinant proteins, protein domains, and in some cases peptides. Generating a vaccine from peptides however requires technologies and concepts very different from classical vaccinology. Immunoinformatics therefore provides the computational tools to propose peptides suitable for formulation into vaccines. This chapter introduces the essential biological concepts affecting design and efficacy of peptide vaccines and discusses current methods and workflows applied to design successful peptide vaccines using computers.

  9. The insulinotropic effect of exogenous glucagon-like peptide-1 is not affected by acute vagotomy in anaesthetized pigs.

    PubMed

    Veedfald, Simon; Hansen, Marie; Christensen, Louise Wulff; Larsen, Sara Agnete Hjort; Hjøllund, Karina Rahr; Plamboeck, Astrid; Hartmann, Bolette; Deacon, Carolyn Fiona; Holst, Jens Juul

    2016-07-01

    What is the central question of this study? We investigated whether intestinal vagal afferents are necessary for the insulinotropic effect of glucagon-like peptide-1 (GLP-1) infused into a mesenteric artery or a peripheral vein before and after acute truncal vagotomy. What is the main finding and its importance? We found no effect of truncal vagotomy on the insulinotropic effect of exogenous GLP-1 and speculate that high circulating concentrations of GLP-1 after i.v. and i.a. infusion might have overshadowed any neural signalling component. We propose that further investigations into the possible vagal afferent signalling of GLP-1 would best be pursued using enteral stimuli to provide high subepithelial levels of endogenous GLP-1. Glucagon-like peptide 1 (GLP-1) is secreted from the gut in response to luminal stimuli and stimulates insulin secretion in a glucose-dependent manner. As a result of rapid enzymatic degradation of GLP-1 by dipeptidyl peptidase-4, a signalling pathway involving activation of intestinal vagal afferents has been proposed. We conducted two series of experiments in α-chloralose-anaesthetized pigs. In protocol I, pigs (n = 14) were allocated for either i.v. or i.a. (mesenteric) GLP-1 infusions (1 and 2 pmol kg(-1)  min(-1) , 30 min) while maintaining permissive glucose concentrations at 6 mmol l(-1) by i.v. glucose infusion. The GLP-1 infusions were repeated after acute truncal vagotomy. In protocol II, pigs (n = 27) were allocated into six groups. Glucagon-like peptide 1 was infused i.v. or i.a. (mesenteric) for 1 h at 3 or 30 pmol kg(-1)  min(-1) . During the steady state (21 min into the GLP-1 infusion), glucose (0.2 g kg(-1) , i.v.) was administered over 9 min to stimulate β-cell secretion. Thirty minutes after the glucose infusion, GLP-1 infusions were discontinued. Following a washout period, the vagal trunks were severed in four of six groups (vagal trunks were left intact in two of six groups), whereupon all

  10. The C-terminal extension peptide of non-photoconvertible water-soluble chlorophyll-binding proteins (Class II WSCPs) affects their solubility and stability: comparative analyses of the biochemical and chlorophyll-binding properties of recombinant Brassica, Raphanus and Lepidium WSCPs with or without their C-terminal extension peptides.

    PubMed

    Takahashi, Shigekazu; Uchida, Akira; Nakayama, Katsumi; Satoh, Hiroyuki

    2014-02-01

    Numerous members of the Brassicaceae possess non-photoconvertible water-soluble chlorophyll (Chl)-binding proteins (Class II WSCPs), which function as Chl scavengers during cell disruption caused by wounding, pest/pathogen attacks, and/or environmental stress. Class II WSCPs have two extension peptides, one at the N-terminus and one at the C-terminus. The N-terminal peptide acts as a signal peptide, targeting the protein to the endoplasmic reticulum body, a unique defensive organelle found only in the Brassicaceae. However, the physiological and biochemical functions of the C-terminal extension peptide had not been characterized previously. To investigate the function of the C-terminal extension peptide, we produced expression constructs of recombinant WSCPs with or without the C-terminal extension peptide. The WSCPs used were of Brussels sprouts (Brassica oleracea), Japanese wild radish (Raphanus sativus) and Virginia pepperweed (Lepidium virginicum). The solubility of all of the WSCPs with the C-terminal extension peptide was drastically lower than that of the recombinant WSCPs without the C-terminal extension peptide. In addition, the stability of the reconstituted WSCPs complexes with the C-terminal extension peptide was altered compared with that of the proteins without the C-terminal extension peptide. These finding indicate that the C-terminal extension peptide affects not only the solubility, but also the stability of Class II WSCP. Furthermore, we characterized the Chl-binding properties of the recombinant WSCP from Japanese wild radish (RshWSCP-His) in a 40 % methanol solution. An electrophoretic mobility shift assay revealed that RshWSCP-His required a half-molar ratio of Chls to form a tetramer.

  11. A new variant in signal peptide of the human luteinizing hormone receptor (LHCGR) affects receptor biogenesis causing leydig cell hypoplasia.

    PubMed

    Vezzoli, Valeria; Duminuco, Paolo; Vottero, Alessandra; Kleinau, Gunnar; Schülein, Ralf; Minari, Roberta; Bassi, Ivan; Bernasconi, Sergio; Persani, Luca; Bonomi, Marco

    2015-11-01

    The human luteinizing hormone/chorionic gonadotropin receptor (LHCGR) plays a fundamental role in male and female reproduction. In males, loss-of-function mutations in LHCGR have been associated with distinct degrees of impairment in pre- and postnatal testosterone secretion resulting in a variable phenotypic spectrum, classified as Leydig cell hypoplasia (LCH) type 1 (complete LH resistance and disorder of sex differentiation) and type 2 (partial LH resistance with impaired masculinization and fertility). Here, we report the case of an adolescent who came to the pediatric endocrinologist at the age of 12 years old for micropenis and cryptorchidism. Testis biopsy showed profound LCH and absent germinal line elements (Sertoli-only syndrome). The sequence analysis of the LHCGR gene showed the presence of a compound heterozygosity, being one variation, c.1847C>A p.S616Y, already described in association to Hypergonadotropic Hypogonadism, and the other, c.29 C>T p.L10P, a new identified variant in the putative signal peptide (SP) of LHCGR. Functional and structural studies provide first evidence that LHCGR have a functional and cleavable SP required for receptor biogenesis. Moreover, we demonstrate the pathogenic role of the novel p.L10P allelic variant, which has to be considered a loss-of-function mutation significantly contributing, in compound heterozygosity with p.S616Y, to the LCH type 2 observed in our patient.

  12. How Identification Processes and Inter-Community Relationships Affect Sense of Community

    ERIC Educational Resources Information Center

    Mannarini, Terri; Rochira, Alessia; Talo, Cosimo

    2012-01-01

    Based on the Social Identity and Social Categorization Theory framework, this study investigated how identification with the physical component of a community (i.e., the place identity), the perception of a community (i.e., the ingroup) in terms of cohesion and entitativity, and the perception of one or more territorial communities as laying…

  13. Identification of avocado (Persea americana) pulp proteins by nano-LC-MS/MS via combinatorial peptide ligand libraries.

    PubMed

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

    2012-09-01

    Avocado (Persea americana) proteins have been scarcely studied despite their importance, especially in food related allergies. The proteome of avocado pulp was explored in depth by extracting proteins with capture by combinatorial peptide ligand libraries at pH 7.4 and under conditions mimicking reverse-phase capture at pH 2.2. The total number of unique gene products identified amounts to 1012 proteins, of which 174 are in common with the control, untreated sample, 190 are present only in the control and 648 represent the new species detected via combinatorial peptide ligand libraries of all combined eluates and likely represent low-abundance proteins. Among the 1012 proteins, it was possible to identify the already known avocado allergen Pers a 1 and different proteins susceptible to be allergens such as a profilin, a polygalacturonase, a thaumatin-like protein, a glucanase, and an isoflavone reductase like protein. PMID:23019098

  14. Identification of avocado (Persea americana) pulp proteins by nano-LC-MS/MS via combinatorial peptide ligand libraries.

    PubMed

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

    2012-09-01

    Avocado (Persea americana) proteins have been scarcely studied despite their importance, especially in food related allergies. The proteome of avocado pulp was explored in depth by extracting proteins with capture by combinatorial peptide ligand libraries at pH 7.4 and under conditions mimicking reverse-phase capture at pH 2.2. The total number of unique gene products identified amounts to 1012 proteins, of which 174 are in common with the control, untreated sample, 190 are present only in the control and 648 represent the new species detected via combinatorial peptide ligand libraries of all combined eluates and likely represent low-abundance proteins. Among the 1012 proteins, it was possible to identify the already known avocado allergen Pers a 1 and different proteins susceptible to be allergens such as a profilin, a polygalacturonase, a thaumatin-like protein, a glucanase, and an isoflavone reductase like protein.

  15. Neutral loss of isocyanic acid in peptide CID spectra: a novel diagnostic marker for mass spectrometric identification of protein citrullination.

    PubMed

    Hao, Gang; Wang, Danchen; Gu, Jane; Shen, Qiuying; Gross, Steven S; Wang, Yanming

    2009-04-01

    Protein citrullination is emerging as an important signaling mechanism that modulates a variety of biological processes. This protein modification constitutes only a 1 Da mass shift, and can be readily confused with other common protein modifications that yield an identical mass shift. In an attempt to develop a robust methodology for detection of protein citrullination sites, we analyzed synthetic citrulline-containing peptides by electrospray ionization tandem mass spectrometry. Collision-induced dissociation (CID) spectra revealed abundant neutral loss of 43 Da from citrullinated peptide precursor ions, which was reconciled by elimination of the HNCO moiety (isocyanic acid) from the citrulline ureido group. The elimination occurs readily in multiple charge states of precursor ions and also in b and y ions. HNCO loss in CID spectra provides a novel diagnostic marker for citrullination, and its utility was demonstrated by the discovery of Arg197 as the specific site of citrullination on nucleophosmin upon peptidylarginine deiminase 4 treatment. PMID:19200748

  16. Identification, expression, and innate immune responses of two insulin-like peptide genes in the razor clam Sinonovacula constricta.

    PubMed

    Niu, Donghong; Wang, Fei; Zhao, Honggang; Wang, Ze; Xie, Shumei; Li, Jiale

    2016-04-01

    Insulin-like peptide (ILP) has emerged as a cell regulatory factor with multiple functions in vertebrates and invertebrates. In the present study, we identified and characterized two ILP genes, ILP1 and ILP2, in the razor clam Sinonovacula constricta. Both ILPs have a signal peptide and a mature domain consisting of six strictly conserved cysteines. The tertiary structure is divided into three main α-helices with a C-domain loop that separates helix 1 from helix 2. Both of ILPs were found to be regulated according to tissue type and developmental stage. After challenge with Vibrio anguillarum, Vibrio parahaemolyticus and Micrococcus lysodeikticus, the expression of two ILP genes was significantly up-regulated in the liver, hemocytes and mantle tissues, suggesting that the ILPs may play roles in the innate immunity in the razor clam Sinonovacula constricta. PMID:26980611

  17. Hybrid in Silico/in Vitro Approach for the Identification of Angiotensin I Converting Enzyme Inhibitory Peptides from Parma Dry-Cured Ham.

    PubMed

    Dellafiora, Luca; Paolella, Sara; Dall'Asta, Chiara; Dossena, Arnaldo; Cozzini, Pietro; Galaverna, Gianni

    2015-07-22

    The bioactivity assessment of foodborne peptides is currently a research area of great relevance, and, in particular, several studies are devoted to the antihypertensive effects through the inhibition of angiotensin I converting enzyme (ACE). In the present work, a straightforward workflow to identify inhibitory peptides from food matrices is proposed, which involves a hybrid in vitro/in silico tandem approach. Parma dry-cured ham was chosen as case study. In particular, the advantage of using the hybrid approach to identify active sequences (in comparison to the experimental trials alone) has been pointed out. Specifically, fractions obtained by in vitro gastrointestinal digestion of ham samples of 18 and 24 months of aging have been assessed for ACE inhibition. At the same time, the released peptidomic profiles, which cannot be entirely evaluated by using in vitro assays, have been screened for the inhibition by using an in silico model. Then, to identify novel inhibitory sequences, a series of strong candidates have been synthesized and assessed for their inhibitory activity through in vitro assay. On the one hand, the use of computational simulations appeared to be an effective strategy to find active sequences, as confirmed by in vitro analysis. On the other hand, strong inhibitory sequences were identified for the first time in Parma dry-cured ham (e.g., LGL and SFVTT with IC50 values of 145 and 395 μM, respectively), which is a product of international dietary and economic relevance. Therefore, these findings demonstrate the usefulness of in silico methodologies coupled to in vitro tests for the identification of potentially bioactive peptides, and they give an important contribution to the study of the overall nutritional value of Parma ham.

  18. Identification of 2D-gel proteins : a comparison of MALDI/TOF peptide mass mapping to {mu} LC-ESI tandem mass spectrometry.

    SciTech Connect

    Lim, H.; Hays, L. G.; Eng, J.; Tollaksen, S. L.; Giometti, C. S.; Holden, J. F.; Adams, M. W. W.; Reich, C. I.; Olsen, G. J.; Yates, J. R.; Biosciences Division; The Scripps Research Inst.; Univ. of Georgia; Univ. of Illinois

    2003-09-01

    A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

  19. Identification of an immunodominant peptide from citrullinated tenascin-C as a major target for autoantibodies in rheumatoid arthritis

    PubMed Central

    Schwenzer, Anja; Jiang, Xia; Mikuls, Ted R; Payne, Jeffrey B; Sayles, Harlan R; Quirke, Anne-Marie; Kessler, Benedikt M; Fischer, Roman; Venables, Patrick J; Lundberg, Karin; Midwood, Kim S

    2016-01-01

    Objectives We investigated whether citrullinated tenascin-C (cTNC), an extracellular matrix protein expressed at high levels in the joints of patients with rheumatoid arthritis (RA), is a target for the autoantibodies in RA. Methods Citrullinated sites were mapped by mass spectrometry in the fibrinogen-like globe (FBG) domain of tenascin-C treated with peptidylarginine deiminases (PAD) 2 and 4. Antibodies to cyclic peptides containing citrullinated sites were screened in sera from patients with RA by ELISA. Potential cross-reactivity with well-established anticitrullinated protein antibody (ACPA) epitopes was tested by inhibition assays. The autoantibody response to one immunodominant cTNC peptide was then analysed in 101 pre-RA sera (median 7 years before onset) and two large independent RA cohorts. Results Nine arginine residues within FBG were citrullinated by PAD2 and PAD4. Two immunodominant peptides cTNC1 (VFLRRKNG-cit-ENFYQNW) and cTNC5 (EHSIQFAEMKL-cit-PSNF-cit-NLEG-cit-cit-KR) were identified. Antibodies to both showed limited cross-reactivity with ACPA epitopes from α-enolase, vimentin and fibrinogen, and no reactivity with citrullinated fibrinogen peptides sharing sequence homology with FBG. cTNC5 antibodies were detected in 18% of pre-RA sera, and in 47% of 1985 Swedish patients with RA and 51% of 287 North American patients with RA. The specificity was 98% compared with 160 healthy controls and 330 patients with osteoarthritis. Conclusions There are multiple citrullination sites in the FBG domain of tenascin-C. Among these, one epitope is recognised by autoantibodies that are detected years before disease onset, and which may serve as a useful biomarker to identify ACPA-positive patients with high sensitivity and specificity in established disease. PMID:26659718

  20. Identification of a Small Peptide That Inhibits PCSK9 Protein Binding to the Low Density Lipoprotein Receptor

    PubMed Central

    Zhang, Yingnan; Eigenbrot, Charles; Zhou, Lijuan; Shia, Steven; Li, Wei; Quan, Clifford; Tom, Jeffrey; Moran, Paul; Di Lello, Paola; Skelton, Nicholas J.; Kong-Beltran, Monica; Peterson, Andrew; Kirchhofer, Daniel

    2014-01-01

    PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 μm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å2 largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 μm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the β-strand and a discontinuous short α-helix, and it engaged in the same β-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9. PMID:24225950

  1. Identification, expression and antibacterial activities of an antimicrobial peptide NK-lysin from a marine fish Larimichthys crocea.

    PubMed

    Zhou, Qi-Jia; Wang, Jun; Liu, Min; Qiao, Ying; Hong, Wan-Shu; Su, Yong-Quan; Han, Kun-Huang; Ke, Qiao-Zhen; Zheng, Wei-Qiang

    2016-08-01

    As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion. PMID:27238427

  2. Identification and Quantification of a New Family of Peptide Endocannabinoids (Pepcans) Showing Negative Allosteric Modulation at CB1 Receptors*

    PubMed Central

    Bauer, Mark; Chicca, Andrea; Tamborrini, Marco; Eisen, David; Lerner, Raissa; Lutz, Beat; Poetz, Oliver; Pluschke, Gerd; Gertsch, Jürg

    2012-01-01

    The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB1). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB1 receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [3H]CP55,940 and [3H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [3H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB1 receptors. Competition binding studies revealed Ki values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [35S]GTPγS binding, and CB1 receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB1 receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling. PMID:22952224

  3. Identification of single base-pair mutation on uidA gene of Escherichia coli O157:H7 by Peptide Nucleic Acids (PNA) mediated PCR clamping.

    PubMed

    Takiya, Toshiyuki; Futo, Satoshi; Tsuna, Mika; Namimatsu, Takanori; Sakano, Tetsuya; Kawai, Keiichi; Suzuki, Tohru

    2004-02-01

    Peptide Nucleic Acids (PNA) is a new type of DNA analogue with a peptide backbone. We developed a rapid identification system of Escherichia. coli O157:H7 using PNA mediated PCR clamping. Firstly, we confirmed a single nucleotide alteration in the uidA gene (T93G), which is specific to E. coli O157: H7. We designed forward mutant DNA primer, wild type PNA, and a reverse DNA primer corresponding to the uidA sequence. PCR cycle consisted of four steps including dual annealing temperatures, 57 degrees C and 45 degrees C. Among 20 E. coli strains with various serotypes and 4 neighboring strains, the amplified bands (517 bp) were detected only in E. coli O157:H7 strains. PNA has specifically inhibited the PCR amplification from a wild type uidA gene. We successfully developed a multiplex PCR system, which detects both shigatoxin (stx) and uidA genes at once, to get reliable results by easier and rapid operation. We also analyzed kinetic parameters of PNA/DNA association using surface plasmon resonance and melting temperature using fluorescence resonance energy transfer (FRET). We discussed a selection mechanism of PCR clamping from these results. PMID:14981299

  4. Characterization and structure identification of an antimicrobial peptide, hominicin, produced by Staphylococcus hominis MBBL 2-9.

    PubMed

    Kim, Pyoung Il; Sohng, Jae Kyung; Sung, Changmin; Joo, Hwang-Soo; Kim, Eun-Mi; Yamaguchi, Tokutaro; Park, Daejoong; Kim, Byung-Gee

    2010-08-20

    Hominicin, antimicrobial peptide displaying potent activity against Staphylococcus aureus ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 11435 and vancomycin-intermediate S. aureus (VISA) CCARM 3501, was purified by chloroform extraction, ion-exchange column chromatography and reverse-phase HPLC from culture supernatant of Staphylococcushominis MBBL 2-9. Hominicin exhibited heat stability up to 121 degrees C for 15min and activity under both acidic and basic conditions (from pH 2.0 to 10.0). Hominicin was cleaved into two fragments after treatment with proteinase K, resulting in the loss of its antibacterial activity, while it was resistant to trypsin, alpha-chymotrypsin, pepsin and lipase. The molecular mass of hominicin determined by mass spectrometry was 2038.4Da. LC-mass spectrometry and NMR spectroscopy analyses of the two fragments revealed the sequence of hominicin as DmIle-Dhb-Pro-Ala-Dhb-Pro-Phe-Dhb-Pro-Ala-Ile-Thr-Glu-Ile-Dhb-Ala-Ala-Val-Ile-Ala-Dmp, which had no similarity with other antimicrobial peptides previously reported. The present study is the first report of this novel antimicrobial peptide, which has uncommon amino acid residues like the ones in Class I group and shows potent activity against clinically relevant S. aureus, MRSA and VISA.

  5. Identification of new, conserved, non-ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine.

    PubMed

    Mossialos, Dimitris; Ochsner, Urs; Baysse, Christine; Chablain, Patrice; Pirnay, Jean-Paul; Koedam, Nico; Budzikiewicz, Herbert; Fernández, Diana Uría; Schäfer, Mathias; Ravel, Jacques; Cornelis, Pierre

    2002-09-01

    Pyoverdines, the main siderophores of fluorescent pseudomonads, contain a peptide moiety, different for each pyoverdine, and an identical chromophore. While it has been shown that non-ribosomal peptide synthetases (NRPSs) are involved in the biosynthesis of the peptide chain of pyoverdines, this was not demonstrated for the biosynthesis of the chromo-phore part. We found that PvsA, from Pseudomonas fluorescens ATCC 17400, and PvdL (PA2424), from Pseudomonas aeruginosa are similar NRPSs and functional homologues, necessary for the production of pyoverdine. Transcriptional lacZ fusions showed that pvdL is co-transcribed with the upstream PA2425 gene, encoding a putative thioesterase, and is iron-regulated via PvdS. Similarly, RT-PCR analysis revealed that expression of pvsA is repressed by iron. Analysis of the adenylation domains of PvsA, PvdL and their homologues, revealed that their N-terminus starts with an acyl-CoA ligase module, followed by three amino acid activation domains. Computer modelling of these domains suggests that PvsA in P. fluorescens and PvdL in P. aeruginosa are orthologues involved in the biosynthesis of the pyoverdine chromophore. PMID:12354233

  6. PEP-SiteFinder: a tool for the blind identification of peptide binding sites on protein surfaces

    PubMed Central

    Saladin, Adrien; Rey, Julien; Thévenet, Pierre; Zacharias, Martin; Moroy, Gautier; Tufféry, Pierre

    2014-01-01

    Peptide–protein interactions are important to many processes of life, particularly for signal transmission or regulatory mechanisms. When no information is known about the interaction between a protein and a peptide, it is of interest to propose candidate sites of interaction at the protein surface, to assist the design of biological experiments to probe the interaction, or to serve as a starting point for more focused in silico approaches. PEP-SiteFinder is a tool that will, given the structure of a protein and the sequence of a peptide, identify protein residues predicted to be at peptide–protein interface. PEP-SiteFinder relies on the 3D de novo generation of peptide conformations given its sequence. These conformations then undergo a fast blind rigid docking on the complete protein surface, and we have found, as the result of a benchmark over 41 complexes, that the best poses overlap to some extent the experimental patch of interaction for close to 90% complexes. In addition, PEP-SiteFinder also returns a propensity index we have found informative about the confidence of the prediction. The PEP-SiteFinder web server is available at http://bioserv.rpbs.univ-paris-diderot.fr/PEP-SiteFinder. PMID:24803671

  7. Identification of protein N-termini in Cyanophora paradoxa cyanelles: transit peptide composition and sequence determinants for precursor maturation

    PubMed Central

    Köhler, Daniel; Dobritzsch, Dirk; Hoehenwarter, Wolfgang; Helm, Stefan; Steiner, Jürgen M.; Baginsky, Sacha

    2015-01-01

    Glaucophyta, rhodophyta, and chloroplastida represent the three main evolutionary lineages that diverged from a common ancestor after primary endosymbiosis. Comparative analyses between members of these three lineages are a rich source of information on ancestral plastid features. We analyzed the composition and the cleavage site of cyanelle transit peptides from the glaucophyte Cyanophora paradoxa by terminal amine labeling of substrates (TAILS), and compared their characteristics to those of representatives of the chloroplastida. Our data show that transit peptide architecture is similar between members of these two lineages. This entails a comparable modular structure, an overrepresentation of serine or alanine and similarities in the amino acid composition around the processing peptidase cleavage site. The most distinctive difference is the overrepresentation of phenylalanine in the N-terminal 1–10 amino acids of cyanelle transit peptides. A quantitative proteome analysis with periplasm-free cyanelles identified 42 out of 262 proteins without the N-terminal phenylalanine, suggesting that the requirement for phenylalanine in the N-terminal region is not absolute. Proteins in this set are on average of low abundance, suggesting that either alternative import pathways are operating specifically for low abundance proteins or that the gene model annotation is incorrect for proteins with fewer EST sequences. We discuss these two possibilities and provide examples for both interpretations. PMID:26257763

  8. Identification of a novel 4 kDa immunoglobulin-A-binding peptide obtained by the limited proteolysis of jacalin.

    PubMed

    Kabir, S; Aebersold, R; Daar, A S

    1993-02-13

    Jacalin, an IgA-binding lectin from jackfruit (Artocarpus heterophyllus) seeds, was isolated by the passage of PBS extracts of seeds over an affinity matrix containing IgA-Sepharose-4B. It was further purified by HPLC. When analyzed by SDS-PAGE under both reducing and nonreducing conditions, the native jacalin was dissociated into two subunits of 12 and 15.4 kDa. Both the subunits could bind IgA. Peptide mapping performed with radioiodinated jacalin indicated that both the subunits were susceptible to proteolysis by Staphylococcus aureus V8 proteinase. One degradation product was a small peptide of 4 kDa. This small proteolytic fragment also bound IgA. The amino-termini of the two major IgA binding subunits, 12 and 15.4 kDa, were identical. The 4 kDa IgA-binding proteolytic fragment of jacalin had a different amino-terminal sequence, suggesting that the region of jacalin which binds IgA does not remain close to the amino-terminus of the peptide.

  9. Stepwise identification of HLA-A*0201-restricted CD8+ T-cell epitope peptides from herpes simplex virus type 1 genome boosted by a StepRank scheme.

    PubMed

    Bi, Jianjun; Song, Rengang; Yang, Huilan; Li, Bingling; Fan, Jianyong; Liu, Zhongrong; Long, Chaoqin

    2011-01-01

    Identification of immunodominant epitopes is the first step in the rational design of peptide vaccines aimed at T-cell immunity. To date, however, it is yet a great challenge for accurately predicting the potent epitope peptides from a pool of large-scale candidates with an efficient manner. In this study, a method that we named StepRank has been developed for the reliable and rapid prediction of binding capabilities/affinities between proteins and genome-wide peptides. In this procedure, instead of single strategy used in most traditional epitope identification algorithms, four steps with different purposes and thus different computational demands are employed in turn to screen the large-scale peptide candidates that are normally generated from, for example, pathogenic genome. The steps 1 and 2 aim at qualitative exclusion of typical nonbinders by using empirical rule and linear statistical approach, while the steps 3 and 4 focus on quantitative examination and prediction of the interaction energy profile and binding affinity of peptide to target protein via quantitative structure-activity relationship (QSAR) and structure-based free energy analysis. We exemplify this method through its application to binding predictions of the peptide segments derived from the 76 known open-reading frames (ORFs) of herpes simplex virus type 1 (HSV-1) genome with or without affinity to human major histocompatibility complex class I (MHC I) molecule HLA-A*0201, and find that the predictive results are well compatible with the classical anchor residue theory and perfectly match for the extended motif pattern of MHC I-binding peptides. The putative epitopes are further confirmed by comparisons with 11 experimentally measured HLA-A*0201-restrcited peptides from the HSV-1 glycoproteins D and K. We expect that this well-designed scheme can be applied in the computational screening of other viral genomes as well. PMID:21072852

  10. Positive relationship between odor identification and affective responses of negatively valenced odors.

    PubMed

    Martinec Nováková, Lenka; Plotěná, Dagmar; Roberts, S Craig; Havlíček, Jan

    2015-01-01

    Hedonic ratings of odors and olfactory preferences are influenced by a number of modulating factors, such as prior experience and knowledge about an odor's identity. The present study addresses the relationship between knowledge about an odor's identity due to prior experience, assessed by means of a test of cued odor identification, and odor pleasantness ratings in children who exhibit ongoing olfactory learning. Ninety-one children aged 8-11 years rated the pleasantness of odors in the Sniffin' Sticks test and, subsequently, took the odor identification test. A positive association between odor identification and pleasantness was found for two unpleasant food odors (garlic and fish): higher pleasantness ratings were exhibited by those participants who correctly identified these odors compared to those who failed to correctly identify them. However, we did not find a similar effect for any of the more pleasant odors. The results of this study suggest that pleasantness ratings of some odors may be modulated by the knowledge of their identity due to prior experience and that this relationship might be more evident in unpleasant odors. PMID:26029143

  11. Positive relationship between odor identification and affective responses of negatively valenced odors

    PubMed Central

    Martinec Nováková, Lenka; Plotěná, Dagmar; Roberts, S. Craig; Havlíček, Jan

    2015-01-01

    Hedonic ratings of odors and olfactory preferences are influenced by a number of modulating factors, such as prior experience and knowledge about an odor’s identity. The present study addresses the relationship between knowledge about an odor’s identity due to prior experience, assessed by means of a test of cued odor identification, and odor pleasantness ratings in children who exhibit ongoing olfactory learning. Ninety-one children aged 8–11 years rated the pleasantness of odors in the Sniffin’ Sticks test and, subsequently, took the odor identification test. A positive association between odor identification and pleasantness was found for two unpleasant food odors (garlic and fish): higher pleasantness ratings were exhibited by those participants who correctly identified these odors compared to those who failed to correctly identify them. However, we did not find a similar effect for any of the more pleasant odors. The results of this study suggest that pleasantness ratings of some odors may be modulated by the knowledge of their identity due to prior experience and that this relationship might be more evident in unpleasant odors. PMID:26029143

  12. Identification, characterization, and synthesis of peptide epitopes and a recombinant six-epitope protein for Trichomonas vaginalis serodiagnosis

    PubMed Central

    Alderete, JF; Neace, Calvin J

    2013-01-01

    There is a need for a rapid, accurate serodiagnostic test useful for both women and men infected by Trichomonas vaginalis, which causes the number one sexually transmitted infection (STI). Women and men exposed to T. vaginalis make serum antibody to fructose-1,6-bisphosphate aldolase (ALD), α-enolase (ENO), and glyceraldehyde-3-phosphate dehydrogenase (GAP). We identified, by epitope mapping, the common and distinct epitopes of each protein detected by the sera of women patients with trichomonosis and by the sera of men highly seropositive to the immunogenic protein α-actinin (positive control sera). We analyzed the amino acid sequences to determine the extent of identity of the epitopes of each protein with other proteins in the databanks. This approach identified epitopes unique to T. vaginalis, indicating these peptide-epitopes as possible targets for a serodiagnostic test. Individual or combinations of 15-mer peptide epitopes with low to no identity with other proteins were reactive with positive control sera from both women and men but were unreactive with negative control sera. These analyses permitted the synthesis of a recombinant His6 fusion protein of 111 amino acids with an Mr of ~13.4 kDa, which consisted of 15-mer peptides of two distinct epitopes each for ALD, ENO, and GAP. This recombinant protein was purified by affinity chromatography. This composite protein was detected by enzyme-linked immunosorbent assay (ELISA), dot blots, and immunoblots, using positive control sera from women and men. These data indicate that it is possible to identify epitopes and that either singly, in combination, or as a composite protein represent targets for a point-of-care serodiagnostic test for T. vaginalis. PMID:27471691

  13. Identification of proopiomelanocortin-related peptides in the rostral pars distalis of the pituitary in coelacanth: evolutional implications.

    PubMed

    Takahashi, Akiyoshi; Yasuda, Akikazu; Sullivan, Craig V; Kawauchi, Hiroshi

    2003-02-15

    The coelacanth fish, genus Latimeria, flourished during the Devonian Period and is considered among the closest living relatives of tetrapods. It may therefore provide important information on the evolution of fishes into tetrapods. However, little is known about the components of the endocrine system in this fish. Here we describe the structural characterization of pituitary hormones derived from proopiomelanocortin (POMC) in Latimeria chalumnae. We identified alpha-melanocyte-stimulating hormone (MSH), N-Des-acetyl-alpha-MSH, beta-MSH, N-terminal peptide containing gamma-MSH, corticotropin-like intermediate lobe peptide (CLIP), and N-acetyl-beta-endorpin (END) in an extract from the rostral pars distalis of the pituitary by reversed-phase high-performance liquid chromatography, amino acid sequence analysis, and mass spectrometry. The occurrence of three different MSHs and one beta-END indicates that the structural organization of coelacanth POMC is the same as that of lungfish, tetrapods, and primitive ray-finned fish. The coelacanth alpha-MSH is identical to its mammalian counterpart. The coelacanth beta-MSH shows the highest sequence identity with the amphibian counterpart, and gamma-MSH and CLIP show the highest sequence identity with their amphibian and bird counterparts, whereas coelacanth beta-END is most similar to the sturgeon peptide. The coexistence of tetrapod-type and fish-type characteristics in the putative coelacanth POMC molecule reflects the phylogenetic position of this fish. When each hormonal segment was compared between coelacanth, lungfish, and tetrapod, MSH and CLIP of coelacanth were closer to their tetrapod counterparts than those of lungfish, whereas beta-MSH and beta-END of coelacanth are less closely related to their tetrapod counterparts than those of lungfish. gamma-MSH and CLIP may have evolved at a different rate from beta-MSH and beta-END in both the coelacanth and lungfish.

  14. Evaluation of Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Identification of Clinically Relevant Mycobacteria in Clinical Specimens and Tissue Sections

    PubMed Central

    Lefmann, Michael; Schweickert, Birgitta; Buchholz, Petra; Göbel, Ulf B.; Ulrichs, Timo; Seiler, Peter; Theegarten, Dirk; Moter, Annette

    2006-01-01

    With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplification-based methods. All isolates (M. tuberculosis complex, n = 24; M. avium, n = 7; M. kansasii, n = 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for species-specific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology. PMID:17021106

  15. Amino acid substitutions in an alpha-helical antimicrobial arachnid peptide affect its chemical properties and biological activity towards pathogenic bacteria but improves its therapeutic index.

    PubMed

    Rodríguez, A; Villegas, E; Satake, H; Possani, L D; Corzo, Gerardo

    2011-01-01

    Four variants of the highly hemolytic antimicrobial peptide Pin2 were chemically synthesized with the aim to investigate the role of the proline residue in this peptide, by replacing it with the motif glycine-valine-glycine [GVG], which was found to confer low hemolytic activity in a spider antimicrobial peptide. The proline residue in position 14 of Pin2 was substituted by [V], [GV], [VG] and [GVG]. Only the peptide variant with the proline substituted for [GVG] was less hemolytic compared to that of all other variants. The peptide variant [GVG] kept its antimicrobial activity in Muller-Hilton agar diffusion assays, whereas the other three variants were less effective. However, all Pin2 antimicrobial peptide variants, were active when challenged against a Gram-positive bacteria in Muller-Hilton broth assays suggesting that chemical properties of the antimicrobial peptides such as hydrophobicity is an important indication for antimicrobial activity in semi-solid environments.

  16. Technology assessment of future intercity passenger transporation systems. Volume 2: Identification of issues affecting intercity transportation

    NASA Technical Reports Server (NTRS)

    1976-01-01

    Papers on major issues and trends that affect the future of intercity transportation are presented. Specific areas covered include: political, social, technological, institutional, and economic mechanisms, the workings of which determine how future intercity transporation technologies will evolve and be put into service; the major issues of intercity transportation from the point of view of reform, including candidate transporation technologies; and technical analysis of trends affecting the evolution of intercity transportation technologies.

  17. Identification of a Potent and Broad-Spectrum Hepatitis C Virus Fusion Inhibitory Peptide from the E2 Stem Domain

    PubMed Central

    Chi, Xiaojing; Niu, Yuqiang; Cheng, Min; Liu, Xiuying; Feng, Yetong; Zheng, Fuxiang; Fan, Jingjing; Li, Xiang; Jin, Qi; Zhong, Jin; Li, Yi-Ping; Yang, Wei

    2016-01-01

    Hepatitis C virus (HCV) envelope proteins E1 and E2 play an essential role in virus entry. However, the fusion mechanisms of HCV remain largely unclear, hampering the development of efficient fusion inhibitors. Here, we developed two cell-based membrane fusion models that allow for screening a peptide library covering the full-length E1 and E2 amino acid sequences. A peptide from the E2 stem domain, named E27, was found to possess the ability to block E1E2-mediated cell-cell fusion and inhibit cell entry of HCV pseudoparticles and infection of cell culture-derived HCV at nanomolar concentrations. E27 demonstrated broad-spectrum inhibition of the major genotypes 1 to 6. A time-of-addition experiment revealed that E27 predominantly functions in the late steps during HCV entry, without influencing the expression and localization of HCV co-receptors. Moreover, we demonstrated that E27 interfered with hetero-dimerization of ectopically expressed E1E2 in cells, and mutational analysis suggested that E27 might target a conserved region in E1. Taken together, our findings provide a novel candidate as well as a strategy for developing potent and broad-spectrum HCV fusion inhibitors, which may complement the current direct-acting antiviral medications for chronic hepatitis C, and shed light on the mechanism of HCV membrane fusion. PMID:27121372

  18. Identification and characterization of Cd-induced peptides in Egeria densa (water weed): Putative role in Cd detoxification.

    PubMed

    Malec, Przemysław; Maleva, Maria G; Prasad, M N V; Strzałka, Kazimierz

    2009-11-27

    Egeria densa has ability to grow in heavy metal contaminated and polluted bodies of water. Shoots exposed to Cd at concentrations up to 300microM for 7 days showed a pronounced decrease in chlorophyll a and in total protein concentration. Thiol-containing compounds and low-molecular-weight polypeptides were detected in Cd-treated plant extracts by gel filtration chromatography. Two Cd-binding fractions, a thiol-enriched fraction and a non-thiol fraction with a lower molecular weight were identified in extracts by gel filtration. The main fraction of thiol-containing polypeptide, purified by gel filtration and anion-exchange chromatography had a molecular weight of approximately 10kDa. This peptide was characterized by a broad absorption band specific to mercaptide bonds and Cd-sensitive fluorescence emission of aromatic amino acid residues. Our results indicate that cadmium exposure of plants resulted in both a formation of thiol-enriched cadmium complexing peptides and a synthesis of low-molecular-weight metal chelators. The putative role of these compounds in Cd detoxification is discussed.

  19. Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments.

    PubMed

    Hanisch, F G; Jovanovic, M; Peter-Katalinic, J

    2001-03-01

    In-gel digestion of densely O-glycosylated proteins, an essential step in proteome analysis, is often hampered by steric hindrance of the proteases. To overcome this technical problem a simple and convenient method has been developed, which combines several advantages: (1) Approximately 70% of the oligosaccharides are cleaved without significant protein hydrolysis at the optimal reaction conditions of 70% ethylamine, and quantitative cleavage is achieved with 40% methylamine, at 50 degrees C. (2) To the unsaturated derivatives of Ser and Thr the alkylamine is added as a label of previous O-glycosylation sites. (3) The alkylaminylated protein is effectively cleaved by proteolysis. (4) The modified peptides are identified by MALDI mass spectrometry under consideration of incremental mass increases. (5) The alkylamine label is stable under MALDI post-source-decay analysis as well as in collision-induced dissociation experiments allowing sequencing and peptide localization of O-glycosylation sites. Applicability of the method is evaluated with a series of synthetic glycopeptides, the densely O-glycosylated human glycophorin A, and with the mucin MUC1 from human milk fat globule membranes.

  20. The effects of shared peptides on protein quantitation in label-free proteomics by LC/MS/MS

    SciTech Connect

    Jin, Shuangshuang; Daly, Don S.; Springer, David L.; Miller, John H.

    2008-01-02

    Assessment of differential protein abundance from the observed properties of detected peptides is an essential part of protein profiling based on shotgun proteomics. However, the abundance observed for degenerate peptides may be due to contributions from multiple proteins that are affected differently by a given treatment. Excluding degenerate peptides eliminates this ambiguity but may significantly decrease the number of proteins for which abundance estimates can be obtained. Peptide degeneracy within a family of biologically related proteins does not cause ambiguity if family members have a common response to treatment. Based on this concept, we have developed an approach for including degenerate peptides in the analysis of differential protein abundance in protein profiling. Data from a recent proteomics study of lung tissue from mice exposed to lipopolysaccharide, cigarette smoke, and a combination of these agents is used to illustrate our method. Starting from data where about half of the protein identifications involved degenerate peptides, 82% of the affected proteins were grouped into families, based on FASTA annotation, with closure on peptide degeneracy. In many cases, a common abundance relative to control was sufficient to explain ion-current peak areas for peptides, both unique and degenerate, that identified biologically-related proteins in a peptide-degeneracy closure group. Based on these results, we propose that peptide-degeneracy closure groups provide a way to include abundance data for degenerate-peptides in quantitative protein profiling by high throughput mass spectrometry.

  1. Subtle Changes in Peptide Conformation Profoundly Affect Recognition of the Non-Classical MHC Class I Molecule HLA-E by the CD94-NKG2 Natural Killer Cell Receptors

    SciTech Connect

    Hoare, Hilary L; Sullivan, Lucy C; Clements, Craig S; Ely, Lauren K; Beddoe, Travis; Henderson, Kate N; Lin, Jie; Reid, Hugh H; Brooks, Andrew G; Rossjohn, Jamie

    2008-03-31

    Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I molecule that binds peptides derived from the leader sequences of other HLA class I molecules. Natural killer cell recognition of these HLA-E molecules, via the CD94-NKG2 natural killer family, represents a central innate mechanism for monitoring major histocompatibility complex expression levels within a cell. The leader sequence-derived peptides bound to HLA-E exhibit very limited polymorphism, yet subtle differences affect the recognition of HLA-E by the CD94-NKG2 receptors. To better understand the basis for this peptide-specific recognition, we determined the structure of HLA-E in complex with two leader peptides, namely, HLA-Cw*07 (VMAPRALLL), which is poorly recognised by CD94-NKG2 receptors, and HLA-G*01 (VMAPRTLFL), a high-affinity ligand of CD94-NKG2 receptors. A comparison of these structures, both of which were determined to 2.5-Å resolution, revealed that allotypic variations in the bound leader sequences do not result in conformational changes in the HLA-E heavy chain, although subtle changes in the conformation of the peptide within the binding groove of HLA-E were evident. Accordingly, our data indicate that the CD94-NKG2 receptors interact with HLA-E in a manner that maximises the ability of the receptors to discriminate between subtle changes in both the sequence and conformation of peptides bound to HLA-E.

  2. Approach/Avoidance Orientations Affect Self-Construal and Identification with In-group

    PubMed Central

    Nussinson, Ravit; Häfner, Michael; Seibt, Beate; Strack, Fritz; Trope, Yaacov

    2011-01-01

    Approach and avoidance are two basic motivational orientations. Their activation influences cognitive and perceptive processes: Previous work suggests that an approach orientation instigates a focus on larger units as compared to avoidance. Study 1 confirms this assumption using a paradigm that more directly taps a person’s tendency to represent objects as belonging to small or large units than prior studies. It was further predicted that the self should also be represented as belonging to larger units, and hence be more interdependent under approach than under avoidance. Study 2 supports this prediction. As a consequence of this focus on belonging to larger units, it was finally predicted that approach results in a stronger identification with one’s in-group than avoidance. Studies 3 and 4 support that prediction. PMID:22844229

  3. Matrix-assisted laser desorption-ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures.

    PubMed

    Lamer, S; Jungblut, P R

    2001-03-10

    In theory, peptide mass fingerprinting by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower microl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.

  4. Peptide vectors for gene delivery: from single peptides to multifunctional peptide nanocarriers.

    PubMed

    Raad, Markus de; Teunissen, Erik A; Mastrobattista, Enrico

    2014-07-01

    The therapeutic use of nucleic acids relies on the availability of sophisticated delivery systems for targeted and intracellular delivery of these molecules. Such a gene delivery should possess essential characteristics to overcome several extracellular and intracellular barriers. Peptides offer an attractive platform for nonviral gene delivery, as several functional peptide classes exist capable of overcoming these barriers. However, none of these functional peptide classes contain all the essential characteristics required to overcome all of the barriers associated with successful gene delivery. Combining functional peptides into multifunctional peptide vectors will be pivotal for improving peptide-based gene delivery systems. By using combinatorial strategies and high-throughput screening, the identification of multifunctional peptide vectors will accelerate the optimization of peptide-based gene delivery systems.

  5. Novel Molecular Assay for Simultaneous Identification of Neglected Lungworms and Heartworms Affecting Cats.

    PubMed

    Di Cesare, Angela; Veronesi, Fabrizia; Frangipane di Regalbono, Antonio; Iorio, Raffaella; Traversa, Donato

    2015-09-01

    Feline lungworms and heartworms are stimulating the interest of the scientific community due to their clinical impact and apparent geographical expansion. Diagnosis of the infections caused by these nematodes is indeed challenging. This report describes a novel multiplex PCR able to identify simultaneously three species of lungworms (Aelurostrongylus abstrusus and Troglostrongylus brevior) and heartworms (Angiostrongylus chabaudi) affecting felids. Epidemiological and clinical perspectives are discussed.

  6. Factors Affecting the Identification of Hispanic English Language Learners in Special Education

    ERIC Educational Resources Information Center

    Becker, Gail I.

    2012-01-01

    This qualitative phenomenological study revealed factors affecting the overrepresentation of Hispanic English language learners (ELLs) in special education. An analysis of the lived experiences of school professionals indicate multiple causes that determine students to be disabled often in violation of state and federal guidelines. Child study…

  7. Identification of EnvC and Its Cognate Amidases as Novel Determinants of Intrinsic Resistance to Cationic Antimicrobial Peptides

    PubMed Central

    Oguri, Tamiko; Yeo, Won-Sik; Bae, Taeok

    2016-01-01

    Cationic antimicrobial peptides (CAMPs) are an essential part of the innate immune system. Some Gram-negative enteric pathogens, such as Salmonella enterica, show intrinsic resistance to CAMPs. However, the molecular basis of intrinsic resistance is poorly understood, largely due to a lack of information about the genes involved. In this study, using a microarray-based genomic technique, we screened the Keio collection of 3,985 Escherichia coli mutants for altered susceptibility to human neutrophil peptide 1 (HNP-1) and identified envC and zapB as novel genetic determinants of intrinsic CAMP resistance. In CAMP killing assays, an E. coli ΔenvCEc or ΔzapBEc mutant displayed a distinct profile of increased susceptibility to both LL-37 and HNP-1. Both mutants, however, displayed wild-type resistance to polymyxin B and human β-defensin 3 (HBD3), suggesting that the intrinsic resistance mediated by EnvC or ZapB is specific to certain CAMPs. A corresponding Salmonella ΔenvCSe mutant showed similarly increased CAMP susceptibility. The envC mutants of both E. coli and S. enterica displayed increased surface negativity and hydrophobicity, which partly explained the increased CAMP susceptibility. However, the ΔenvCEc mutant, but not the ΔenvCSe mutant, was defective in outer membrane permeability, excluding this defect as a common factor contributing to the increased CAMP susceptibility. Animal experiments showed that the Salmonella ΔenvCSe mutant had attenuated virulence. Taken together, our results indicate that the role of envC in intrinsic CAMP resistance is likely conserved among Gram-negative enteric bacteria, demonstrate the importance of intrinsic CAMP resistance for full virulence of S. enterica, and provide insight into distinct mechanisms of action of CAMPs. PMID:26810659

  8. Antiplatelet Aggregation and Antithrombosis Efficiency of Peptides in the Snake Venom of Deinagkistrodon acutus: Isolation, Identification, and Evaluation

    PubMed Central

    Ding, Bin; Xu, Zhenghong; Qian, Chaodong; Jiang, Fusheng; Ding, Xinghong; Ruan, Yeping; Ding, Zhishan; Fan, Yongsheng

    2015-01-01

    Two peptides of Pt-A (Glu-Asn-Trp 429 Da) and Pt-B (Glu-Gln-Trp 443 Da) were isolated from venom liquor of Deinagkistrodon acutus. Their antiplatelet aggregation effects were evaluated with platelet-rich human plasma in vitro; the respective IC50 of Pt-A and Pt-B was 66 μM and 203 μM. Both peptides exhibited protection effects on ADP-induced paralysis in mice. After ADP administration, the paralysis time of different concentration of Pt-A and Pt-B lasted as the following: 80 mg/kg Pt-B (152.8 ± 57.8 s) < 40 mg/kg Pt-A (163.5 ± 59.8 s) < 20 mg/kg Pt-A (253.5 ± 74.5 s) < 4 mg/kg clopidogrel (a positive control, 254.5 ± 41.97 s) < 40 mg/kg Pt-B (400.8 ± 35.9 s) < 10 mg/kg Pt-A (422.8 ± 55.4 s), all of which were statistically shorter than the saline treatment (666 ± 28 s). Pulmonary tissue biopsy confirmed that Pt-A and Pt-B prevented the formation of thrombi in the lung. Unlike ADP injection alone, which caused significant reduction of peripheral platelet count, Pt-A treatment prevented the drop of peripheral platelet counts; interestingly, Pt-B could not, even though the same amount of Pt-B also showed protection effects on ADP-induced paralysis and thrombosis. More importantly, intravenous injection of Pt-A and Pt-B did not significantly increase the hemorrhage risks as clopidogrel. PMID:26483843

  9. Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies

    DOE PAGES

    Donaldson, Joshua M.; Zer, Cindy; Avery, Kendra N.; Bzymek, Krzysztof P.; Horne, David A.; Williams, John C.

    2013-10-07

    Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximabmore » (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Lastly, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent “linker” for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.« less

  10. Identification of multiple peptides with antioxidant and antimicrobial activities from skin and its secretions of Hylarana taipehensis, Amolops lifanensis, and Amolops granulosus.

    PubMed

    Guo, Chao; Hu, Yuhong; Li, Jing; Liu, Yuliang; Li, Sihan; Yan, Keqiang; Wang, Xiao; Liu, Jingze; Wang, Hui

    2014-10-01

    Amphibian skin and its secretions contain many kinds of peptides with different bioactivities. In this study, a large number of peptides including antioxidant and antimicrobial peptides were identified from three East Asian frog species Hylarana taipehensis, Amolops lifanensis, and Amolops granulosus. The majority of these peptides were antimicrobial peptides, while eight antioxidant peptides were identified, which included two novel peptides taipehensin-1TP1 (TLIWEFYHQILDEYNKENKG) and taipehensin-2TP1 (CLMARPNYRCKIFKQC). These antioxidant peptides exhibited the ability to scavenge ABTS and/or DPPH free radicals. Moreover, six out of eight antioxidant peptides temporin-TP1, brevinin-1TP1, brevinin-1TP2, brevinin-1TP3, brevinin-1LF1, and palustrin-2GN1 also showed antimicrobial activity.

  11. Cyclotide Discovery in Gentianales Revisited—Identification and Characterization of Cyclic Cystine-Knot Peptides and Their Phylogenetic Distribution in Rubiaceae Plants

    PubMed Central

    Koehbach, Johannes; Attah, Alfred F.; Berger, Andreas; Hellinger, Roland; Kutchan, Toni M.; Carpenter, Eric J.; Rolf, Megan; Sonibare, Mubo A.; Moody, Jones O.; Ka-Shu Wong, Gane; Dessein, Steven; Greger, Harald; Gruber, Christian W.

    2013-01-01

    Cyclotides are a unique class of ribosomally synthesized cysteine-rich miniproteins characterized by a head-to-tail cyclized backbone and three conserved disulfide-bonds in a knotted arrangement. Originally they were discovered in the coffee-family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide-containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sampling numbers. As a consequence, their phylogeny in the plant kingdom remains unclear. To gain further insight into the distribution and evolution of plant cyclotides, we analyzed ~300 species of >40 different families, with special emphasis on plants from the order Gentianales. For this purpose, we have developed a refined screening methodology combining chemical analysis of plant extracts and bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome-mining, we identified nine novel cyclotide-containing species and their related cyclotide precursor genes in the tribe Palicoureeae. The characterization of novel peptide sequences underlines the high variability and plasticity of the cyclotide framework, and a comparison of novel precursor proteins from Carapichea ipecacuanha illustrated their typical cyclotide gene architectures. Phylogenetic analysis of their distribution within the Psychotria alliance revealed cyclotides to be restricted to Palicourea, Margaritopsis, Notopleura, Carapichea, Chassalia, and Geophila. In line with previous reports, our findings confirm cyclotides to be one of the largest peptide families within the plant kingdom and suggest that their total number may exceed tens of thousands. PMID:23897543

  12. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

    PubMed

    Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2016-01-01

    Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy. PMID:26942714

  13. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Deliv