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Sample records for random peptide libraries

  1. ATP selection in a random peptide library consisting of prebiotic amino acids.

    PubMed

    Kang, Shou-Kai; Chen, Bai-Xue; Tian, Tian; Jia, Xi-Shuai; Chu, Xin-Yi; Liu, Rong; Dong, Peng-Fei; Yang, Qing-Yong; Zhang, Hong-Yu

    2015-10-23

    Based upon many theoretical findings on protein evolution, we proposed a ligand-selection model for the origin of proteins, in which the most ancient proteins originated from ATP selection in a pool of random peptides. To test this ligand-selection model, we constructed a random peptide library consisting of 15 types of prebiotic amino acids and then used cDNA display to perform six rounds of in vitro selection with ATP. By means of next-generation sequencing, the most prevalent sequence was defined. Biochemical and biophysical characterization of the selected peptide showed that it was stable and foldable and had ATP-hydrolysis activity as well.

  2. Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries

    PubMed Central

    Yang, Li-Min; Wang, Jing-Lin; Kang, Lin; Gao, Shan; Liu, Yan-hua; Hu, Ting-Mao

    2008-01-01

    Random peptide libraries displayed on the ribosome are becoming a new tool for the in vitro selection of biologically relevant macromolecules, including epitopes, antagonists, enzymes, and cell-surface receptors. Ribosome display is a cell-free system of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes) by the formation of stable protein-ribosome-mRNA complexes and permitting the selection of a functional nascent protein by iterative cycles of panning and reverse transcription-polymerase chain reaction (RT-PCR) amplification in vitro. The complexity of the random peptide library is critical for the success of a panning experiment; greater the diversity of sequences within the library, the more likely it is that the library comprises sequences that can bind a given target with specific affinity. Here, we have used the cell-free system Escherichia coli S30 lysate to construct high-complexity random peptide libraries (>1014 independent members) by introducing strategies that are different from the methods described by Mattheakis et al. and Lamla et al. The key step in our method is to produce nanomole (nmol) amounts of DNA elements that are necessary for in vitro transcription/translation by using PCR but not plasmid DNA. Library design strategies and protocols that facilitate rapid identification are also presented. PMID:18493302

  3. Human IgA-binding peptides selected from random peptide libraries: affinity maturation and application in IgA purification.

    PubMed

    Hatanaka, Takaaki; Ohzono, Shinji; Park, Mirae; Sakamoto, Kotaro; Tsukamoto, Shogo; Sugita, Ryohei; Ishitobi, Hiroyuki; Mori, Toshiyuki; Ito, Osamu; Sorajo, Koichi; Sugimura, Kazuhisa; Ham, Sihyun; Ito, Yuji

    2012-12-14

    Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1-A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (K(d) = 1.3 μm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (K(d) = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.

  4. Isolation of peptides from phage-displayed random peptide libraries that interact with the talin-binding domain of vinculin.

    PubMed Central

    Adey, N B; Kay, B K

    1997-01-01

    Peptides isolated from combinatorial libraries typically interact with, and thus help to characterize, biologically relevant binding domains of target proteins. To characterize the binding domains of the focal adhesion protein vinculin, vinculin-binding peptides were isolated from two phage-displayed random peptide libraries. Altogether, five non-similar vinculin-binding peptides were identified. Despite the lack of obvious sequence similarity between the peptides, binding and competition studies indicated that all five interact with the talin-binding domain of vinculin and do not disrupt the binding of alpha-actinin or paxillin to vinculin. The identified peptides and talin bind to vinculin in a comparable manner; both bind to immobilized vinculin, but neither binds to soluble vinculin unless the C-terminus of vinculin has been deleted. An analysis of amino acid variants of one of the peptides has revealed three non-contiguous motifs that also occur in the region of talin previously demonstrated to bind vinculin. Amino acid substitutions within a 127-residue segment of talin capable of binding vinculin confirmed the importance of two of the motifs and suggest that residues critical for binding are within a 16-residue region. This study demonstrates that the vinculin-binding peptides interact with vinculin in a biologically relevant manner and represent an excellent tool for further study of the biochemistry of vinculin. PMID:9182713

  5. Characterization of Seven New Polystyrene Plates Binding Peptides from a Phage-Displayed Random 12-Peptide Library.

    PubMed

    Hu, Yun-Fei; Gao, Xiao-Chen; Xu, Tian-Qi; Dun, Zhao; Yu, Xing-Long

    2016-01-01

    A random 12-peptide library was screened against Erysipelothrix rhusiopthiae and porcine circovirus 2 recombinant Cap protein and the selected peptides were used for detecting the corresponding pathogens quickly and effectively. To our surprise, seven peptides, P1 (WHWNAP WWNGVY), P2 (FHWTWQFPYTST), P3 (GAMHLPWHMGTL), P4 (HWNIWWQHHPSP), P5 (HFFKWHTRTNDQ), P6 (HFFRWHPSAHLG) and P7 (HFAYWWNGVRGP) with the characteristics of polystyrene plate (PS) binding target-unrelated peptides (TUPs), were selected from the library. It has been found that P2 and P4 shared common motif of plastic binding peptide, moreover, P2, P3, P5 and P7 have been isolated repeatedly in other research groups using different targets. Then, the seven peptide phage clones were identified as the PS binding TUP phages by phage-ELISA and elution titration, particularly, P1 and P2 showed strong PS binding affinity which can not be inhibited by usual blocking buffers. In addition, all of the phages were not propagation-related TUP, but P3 showed the similar propagation rate with M13KE (vector phage). We also found that the seven PS-TUPs are rich in W, H, F, P and G, particularly, both W and H are contained in all PS-TUPs. It deduced that they may play a potential role in peptide binding to plastic. Although it is difficult to eliminate the TUP phages in phage display completely, these PS-TUPs can be used to exclude the false positive peptides rapidly and effectively and help us to obtain truly interesting peptides more accurately. PMID:26980286

  6. 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

  7. 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.

  8. Multiplexed Random Peptide Library and Phospho-Specific Antibodies Facilitate Human Polo-Like Kinase 1 Inhibitor Screen

    PubMed Central

    Koresawa, Mitsunori; Iida, Masato; Fukasawa, Kazuhiro; Stec, Erica; Cassaday, Jason; Chase, Peter; Rickert, Keith; Hodder, Peter; Takagi, Toshimitsu; Komatani, Hideya

    2010-01-01

    Abstract One of the challenges to develop time-resolved fluorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/Thr) protein kinase is to select an optimal peptide substrate and a specific phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1). A screen of a diverse peptide library in a 384-well plate format identified several highly potent substrates that share the consensus motif for phosphorylation by Plk1. Their potencies were comparable to FKD peptide, a designed peptide substrate derived from well-described Plk1 substrate Cdc25C. A specific anti-phosphor Ser/Thr antibody p(S/T)F antibody that detects the phosphorylation of FKD peptide was screened out of 87 antibodies with time-resolved fluorometry technology in a 96-well plate format. Using FKD peptide and p(S/T)F antibody, we successfully developed a robust TR-FRET assay in 384-well plate format, and further miniaturized this assay to 1,536-well plate format to perform uHTS. We screened about 1.2 million compounds for Plk1 inhibitors using a Plk1 deletion mutant that only has the kinase domain and subsequently screened the same compound library using a full-length active-mutant Plk1. These uHTSs identified a number of hit compounds, and some of them had selectivity to either the deletion mutant or the full-length protein. Our results prove that a combination of random screen for substrate peptide and phospho-specific antibodies is very powerful strategy to develop TR-FRET assays for protein kinases. PMID:20085455

  9. Development of a novel efficient method to construct an adenovirus library displaying random peptides on the fiber knob

    PubMed Central

    Yamamoto, Yuki; Goto, Naoko; Miura, Kazuki; Narumi, Kenta; Ohnami, Shumpei; Uchida, Hiroaki; Miura, Yoshiaki; Yamamoto, Masato; Aoki, Kazunori

    2014-01-01

    Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 104 level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:24380399

  10. Development of a novel efficient method to construct an adenovirus library displaying random peptides on the fiber knob.

    PubMed

    Yamamoto, Yuki; Goto, Naoko; Miura, Kazuki; Narumi, Kenta; Ohnami, Shumpei; Uchida, Hiroaki; Miura, Yoshiaki; Yamamoto, Masato; Aoki, Kazunori

    2014-03-01

    Redirection of adenovirus vectors by engineering the capsid-coding region has shown limited success because proper targeting ligands are generally unknown. To overcome this limitation, we constructed an adenovirus library displaying random peptides on the fiber knob, and its screening led to successful selections of several particular targeted vectors. In the previous library construction method, the full length of an adenoviral genome was generated by a Cre-lox mediated in vitro recombination between a fiber-modified plasmid library and the enzyme-digested adenoviral DNA/terminal protein complex (DNA-TPC) before transfection to the producer cells. In this system, the procedures were complicated and time-consuming, and approximately 30% of the vectors in the library were defective with no displaying peptide. These may hinder further extensive exploration of cancer-targeting vectors. To resolve these problems, in this study, we developed a novel method with the transfection of a fiber-modified plasmid library and a fiberless adenoviral DNA-TPC in Cre-expressing 293 cells. The use of in-cell Cre recombination and fiberless adenovirus greatly simplified the library-making steps. The fiberless adenovirus was useful in suppressing the expansion of unnecessary adenovirus vectors. In addition, the complexity of the library was more than a 10(4) level in one well in a 6-well dish, which was 10-fold higher than that of the original method. The results demonstrated that this novel method is useful in producing a high quality live adenovirus library, which could facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:24380399

  11. Novel β-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy

    PubMed Central

    Shukla, Girja S.; Krag, David N.

    2010-01-01

    Novel phage-displayed random linear dodecapeptide (X12) and cysteine-constrained decapeptide (CX10C) libraries constructed in fusion to the amino-terminus of P99 β-lactamase molecules were used for identifying β-lactamase-linked cancer cell-specific ligands. The size and quality of both libraries were comparable to the standards of other reported phage display systems. Using the single-round panning method based on phage DNA recovery, we identified severalβ-lactamase fusion peptides that specifically bind to live human breast cancer MDA-MB-361 cells. The β-lactamase fusion to the peptides helped in conducting the enzyme activity-based clone normalization and cell-binding screening in a very time- and cost-efficient manner. The methods were suitable for 96-well readout as well as microscopic imaging. The success of the biopanning was indicated by the presence of ~40% cancer cell-specific clones among recovered phages. One of the binding clones appeared multiple times. The cancer cell-binding fusion peptides also shared several significant motifs. This opens a new way of preparing and selecting phage display libraries. The cancer cell-specific β-lactamase-linked affinity reagents selected from these libraries can be used for any application that requires a reporter for tracking the ligand molecules. Furthermore, these affinity reagents have also a potential for their direct use in the targeted enzyme prodrug therapy of cancer. PMID:19751096

  12. Screening of random peptide library of hemagglutinin from pandemic 2009 A(H1N1) influenza virus reveals unexpected antigenically important regions.

    PubMed

    Xu, Wanghui; Han, Lu; Lin, Zhanglin

    2011-01-01

    The antigenic structure of the membrane protein hemagglutinin (HA) from the 2009 A(H1N1) influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS) against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify "hot spots" or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins. PMID:21437206

  13. Identification of a Conserved Linear B-Cell Epitope of Streptococcus dysgalactiae GapC Protein by Screening Phage-Displayed Random Peptide Library

    PubMed Central

    Fan, Ziyao; Zhou, Xue; Yu, Liquan; Sun, Hunan; Wu, Zhijun; Yu, Yongzhong; Song, Baifen; Ma, Jinzhu; Tong, Chunyu; Wang, Xintong; Zhu, Zhanbo; Cui, Yudong

    2015-01-01

    The GapC of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce protective humoral immune response in animals. However, B-cell epitopes on the S. dysgalactiae GapC have not been well identified. In this study, a monoclonal antibody (mAb5B7) against the GapC1-150 protein was prepared. After passive transfer, mAb5B7 could partially protect mice against S. dysgalactiae infection. Eleven positive phage clones recognized by mAb5B7 were identified by screening phage-displayed random 12-peptide library, most of which matched the consensus motif DTTQGRFD. The motif sequence exactly matches amino acids 48-55 of the S. dysgalactiae GapC protein. In addition, the motif 48DTTQGRFD55 shows high homology among various streptococcus species. Site-directed mutagenic analysis further confirmed that residues D48, T50, Q51, G52 and F54 formed the core motif of 48DTTQGRFD55. This motif was the minimal determinant of the B-cell epitope recognized by the mAb5B7. As expected, epitope-peptide evoked protective immune response against S. dysgalactiae infection in immunized mice. Taken together, this identified conserved B-cell epitope within S. dysgalactiae GapC could provide very valuable insights for vaccine design against S. dysgalactiae infection. PMID:26121648

  14. Biomathematical description of synthetic peptide libraries.

    PubMed

    Sieber, Timo; Hare, Eric; Hofmann, Heike; Trepel, Martin

    2015-01-01

    Libraries of randomised peptides displayed on phages or viral particles are essential tools in a wide spectrum of applications. However, there is only limited understanding of a library's fundamental dynamics and the influences of encoding schemes and sizes on their quality. Numeric properties of libraries, such as the expected number of different peptides and the library's coverage, have long been in use as measures of a library's quality. Here, we present a graphical framework of these measures together with a library's relative efficiency to help to describe libraries in enough detail for researchers to plan new experiments in a more informed manner. In particular, these values allow us to answer-in a probabilistic fashion-the question of whether a specific library does indeed contain one of the "best" possible peptides. The framework is implemented in a web-interface based on two packages, discreteRV and peptider, to the statistical software environment R. We further provide a user-friendly web-interface called PeLiCa (Peptide Library Calculator, http://www.pelica.org), allowing scientists to plan and analyse their peptide libraries. PMID:26042419

  15. Biomathematical description of synthetic peptide libraries.

    PubMed

    Sieber, Timo; Hare, Eric; Hofmann, Heike; Trepel, Martin

    2015-01-01

    Libraries of randomised peptides displayed on phages or viral particles are essential tools in a wide spectrum of applications. However, there is only limited understanding of a library's fundamental dynamics and the influences of encoding schemes and sizes on their quality. Numeric properties of libraries, such as the expected number of different peptides and the library's coverage, have long been in use as measures of a library's quality. Here, we present a graphical framework of these measures together with a library's relative efficiency to help to describe libraries in enough detail for researchers to plan new experiments in a more informed manner. In particular, these values allow us to answer-in a probabilistic fashion-the question of whether a specific library does indeed contain one of the "best" possible peptides. The framework is implemented in a web-interface based on two packages, discreteRV and peptider, to the statistical software environment R. We further provide a user-friendly web-interface called PeLiCa (Peptide Library Calculator, http://www.pelica.org), allowing scientists to plan and analyse their peptide libraries.

  16. Peptide and peptide library cyclization via bromomethylbenzene derivatives.

    PubMed

    Hacker, David E; Almohaini, Mohammed; Anbazhagan, Aruna; Ma, Zhong; Hartman, Matthew C T

    2015-01-01

    Cyclization confers several advantages to peptides, cumulatively serving to make them more drug-like. In this protocol, cyclic peptides are generated via bis-alkylation of cysteine-containing peptides using α,α'-dibromo-m-xylene. The reactions are robust and high yielding. Multiple reaction platforms for the application of this versatile strategy are described herein: the cyclization of solid-phase-synthesized peptides, both in solution and on resin, as well as the cyclization of in vitro translated mRNA-peptide fusion libraries on oligo(dT) resin.

  17. Potential of phage-displayed peptide library technology to identify functional targeting peptides

    PubMed Central

    Krumpe, Lauren RH; Mori, Toshiyuki

    2010-01-01

    Combinatorial peptide library technology is a valuable resource for drug discovery and development. Several peptide drugs developed through phage-displayed peptide library technology are presently in clinical trials and the authors envision that phage-displayed peptide library technology will assist in the discovery and development of many more. This review attempts to compile and summarize recent literature on targeting peptides developed through peptide library technology, with special emphasis on novel peptides with targeting capacity evaluated in vivo. PMID:20150977

  18. Encoded libraries of chemically modified peptides.

    PubMed

    Heinis, Christian; Winter, Greg

    2015-06-01

    The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries.

  19. Implicit biology in peptide spectral libraries.

    PubMed

    Askenazi, Manor; Linial, Michal

    2012-09-18

    Mass spectral libraries are collections of mass spectra curated specifically to facilitate the identification of small molecules, metabolites, and short peptides. One of the most comprehensive peptide spectral libraries is curated by NIST and contains upward of half a million annotated spectra dominated by human and model organisms including budding yeast and mouse. While motivated primarily by the technological goal of increasing sensitivity and specificity in spectral identification, we have found that the NIST spectral library constitutes a surprisingly rich source of biological knowledge. In this Article, we show that data-mining of these published libraries while applying strict empirical thresholds yields many characteristics of protein biology. In particular, we demonstrate that the size and increasingly comprehensive nature of these libraries, generated from whole-proteome digests, enables inference from the presence but crucially also from the absence of spectra for individual peptides. We illustrate implicit biological trends that lead to significant absence of spectra accounted for by complex post-translational modifications and overlooked proteolytic sites. We conclude that many subtle biological signatures such as genetic variants, regulated proteolysis, and post-translational modifications are exposed through the systematic mining of spectral collections originally compiled as general-purpose, technology-oriented resources.

  20. Selection dynamic of Escherichia coli host in M13 combinatorial peptide phage display libraries.

    PubMed

    Zanconato, Stefano; Minervini, Giovanni; Poli, Irene; De Lucrezia, Davide

    2011-01-01

    Phage display relies on an iterative cycle of selection and amplification of random combinatorial libraries to enrich the initial population of those peptides that satisfy a priori chosen criteria. The effectiveness of any phage display protocol depends directly on library amino acid sequence diversity and the strength of the selection procedure. In this study we monitored the dynamics of the selective pressure exerted by the host organism on a random peptide library in the absence of any additional selection pressure. The results indicate that sequence censorship exerted by Escherichia coli dramatically reduces library diversity and can significantly impair phage display effectiveness. PMID:21512219

  1. 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

  2. PASSEL: the PeptideAtlas SRMexperiment library.

    PubMed

    Farrah, Terry; Deutsch, Eric W; Kreisberg, Richard; Sun, Zhi; Campbell, David S; Mendoza, Luis; Kusebauch, Ulrike; Brusniak, Mi-Youn; Hüttenhain, Ruth; Schiess, Ralph; Selevsek, Nathalie; Aebersold, Ruedi; Moritz, Robert L

    2012-04-01

    Public repositories for proteomics data have accelerated proteomics research by enabling more efficient cross-analyses of datasets, supporting the creation of protein and peptide compendia of experimental results, supporting the development and testing of new software tools, and facilitating the manuscript review process. The repositories available to date have been designed to accommodate either shotgun experiments or generic proteomic data files. Here, we describe a new kind of proteomic data repository for the collection and representation of data from selected reaction monitoring (SRM) measurements. The PeptideAtlas SRM Experiment Library (PASSEL) allows researchers to easily submit proteomic data sets generated by SRM. The raw data are automatically processed in a uniform manner and the results are stored in a database, where they may be downloaded or browsed via a web interface that includes a chromatogram viewer. PASSELenables cross-analysis of SRMdata, supports optimization of SRMdata collection, and facilitates the review process of SRMdata. Further, PASSELwill help in the assessment of proteotypic peptide performance in a wide array of samples containing the same peptide, as well as across multiple experimental protocols.

  3. Spectral Library Searching To Identify Cross-Linked Peptides.

    PubMed

    Schweppe, Devin K; Chavez, Juan D; Navare, Arti T; Wu, Xia; Ruiz, Bianca; Eng, Jimmy K; Lam, Henry; Bruce, James E

    2016-05-01

    Methods harnessing protein cross-linking and mass spectrometry (XL-MS) offer high-throughput means to identify protein-protein interactions (PPIs) and structural interfaces of protein complexes. Yet, specialized data dependent methods and search algorithms are often required to confidently assign peptide identifications to spectra. To improve the efficiency of matching high confidence spectra, we developed a spectral library based approach to search cross-linked peptide data derived from Protein Interaction Reporter (PIR) methods using the spectral library search algorithm, SpectraST. Spectral library matching of cross-linked peptide data from query spectra increased the absolute number of confident peptide relationships matched to spectra and thereby the number of PPIs identified. By matching library spectra from bona fide, previously established PIR-cross-linked peptide relationships, spectral library searching reduces the need for continued, complex mass spectrometric methods to identify peptide relationships, increases coverage of relationship identifications, and improves the accessibility of XL-MS technologies.

  4. Calculating complexity of large randomized libraries.

    PubMed

    Kong, Yong

    2009-08-01

    Randomized libraries are increasingly popular in protein engineering and other biomedical research fields. Statistics of the libraries are useful to guide and evaluate randomized library construction. Previous works only give the mean of the number of unique sequences in the library, and they can only handle equal molar ratio of the four nucleotides at a small number of mutation sites. We derive formulas to calculate the mean and variance of the number of unique sequences in libraries generated by cassette mutagenesis with mixtures of arbitrary nucleotide ratios. Computer program was developed which utilizes arbitrary numerical precision software package to calculate the statistics of large libraries. The statistics of library with mutations in more than 20 amino acids can be calculated easily. Results show that the nucleotide ratios have significant effects on these statistics. The more skewed the ratio, the larger the library size is needed to obtain the same expected number of unique sequences. The program is freely available at http://graphics.med.yale.edu/cgi-bin/lib_comp.pl.

  5. 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).

  6. Combinatorial Library of Improved Peptide Aptamers, CLIPs to Inhibit RAGE Signal Transduction in Mammalian Cells

    PubMed Central

    Reverdatto, Sergey; Rai, Vivek; Xue, Jing; Burz, David S.; Schmidt, Ann Marie; Shekhtman, Alexander

    2013-01-01

    Peptide aptamers are small proteins containing a randomized peptide sequence embedded into a stable protein scaffold, such as Thioredoxin. We developed a robust method for building a Combinatorial Library of Improved Peptide aptamers (CLIPs) of high complexity, containing ≥3×1010 independent clones, to be used as a molecular tool in the study of biological pathways. The Thioredoxin scaffold was modified to increase solubility and eliminate aggregation of the peptide aptamers. The CLIPs was used in a yeast two-hybrid screen to identify peptide aptamers that bind to various domains of the Receptor for Advanced Glycation End products (RAGE). NMR spectroscopy was used to identify interaction surfaces between the peptide aptamers and RAGE domains. Cellular functional assays revealed that in addition to directly interfering with known binding sites, peptide aptamer binding distal to ligand sites also inhibits RAGE ligand-induced signal transduction. This finding underscores the potential of using CLIPs to select allosteric inhibitors of biological targets. PMID:23785412

  7. Peptides on phage: a vast library of peptides for identifying ligands.

    PubMed Central

    Cwirla, S E; Peters, E A; Barrett, R W; Dower, W J

    1990-01-01

    We have constructed a vast library of peptides for finding compounds that bind to antibodies and other receptors. Millions of different hexapeptides were expressed at the N terminus of the adsorption protein (pIII) of fd phage. The vector fAFF1, derived from the tetracycline resistance-transducing vector fd-tet, allows cloning of oligonucleotides in a variety of locations in the 5' region of gene III. A library of 3 x 10(8) recombinants was generated by cloning randomly synthesized oligonucleotides. The library was screened for high-avidity binding to a monoclonal antibody (3-E7) that is specific for the N terminus of beta-endorphin (Tyr-Gly-Gly-Phe). Fifty-one clones selected by three rounds of the affinity purification technique called panning were sequenced and found to differ from previously known ligands for this antibody. The striking finding is that all 51 contained tyrosine as the N-terminal residue and that 48 contained glycine as the second residue. The binding affinities of six chemically synthesized hexapeptides from this set range from 0.35 microM (Tyr-Gly-Phe-Trp-Gly-Met) to 8.3 microM (Tyr-Ala-Gly-Phe-Ala-Gln), compared with 7.1 nM for a known high-affinity ligand (Tyr-Gly-Gly-Phe-Leu). These results show that ligands can be identified with no prior information concerning antibody specificity. Peptide libraries are also likely to be useful in finding ligands that bind to other classes of receptors and in discovering pharmacologic agents. Images PMID:2201029

  8. Random Glycopeptide Bead Libraries for Seromic Biomarker Discovery

    PubMed Central

    Kračun, Stjepan K.; Cló, Emilano; Clausen, Henrik; Levery, Steven B.; Jensen, Knud J.; Blixt, Ola

    2010-01-01

    Identification of disease specific biomarkers is important to address early diagnosis and management of disease. Aberrant post-translational modifications (PTM) of proteins such as O-glycosylations (O-PTMs) are emerging as triggers of autoantibodies that can serve as sensitive biomarkers. Here we have developed a random glycopeptide bead library screening platform for detection of autoantibodies and other binding proteins. Libraries were build on biocompatible PEGA beads including a safety-catch C-terminal amide linker (SCAL) that allowed mild cleavage conditions (I2/NaBH4 and TFA) for release of glycopeptides and sequence determination by ESI-MSn. As proof-of principle, tumor specific glycopeptide reporter epitopes were built-in into the libraries and were detected by tumor specific monoclonal antibodies and autoantibodies from cancer patients. Sequenced and identified glycopeptides were re-synthesized at preparative scale by automated parallel peptide synthesis and printed on microarrays for validation and broader analysis with larger sets of sera. We further showed that chemical synthesis of the monosaccharide O-glycopeptide library (Tn-glycoform) could be diversified to other tumor glycoforms by on-bead enzymatic glycosylation reactions with recombinant glycosyltransferases. Hence, we have developed a high-throughput flexible platform for rapid biomarker discovery O-glycopeptides and the method has applicability in other types of assays like lectin/antibody/enzyme specificity studies as well as investigation of other PTMs. PMID:20886906

  9. An overhang-based DNA block shuffling method for creating a customized random library

    PubMed Central

    Fujishima, Kosuke; Venter, Chris; Wang, Kendrick; Ferreira, Raphael; Rothschild, Lynn J.

    2015-01-01

    We present an overhang-based DNA block shuffling method to create a customized random DNA library with flexible sequence design and length. Our method enables the efficient and seamless assembly of short DNA blocks with dinucleotide overhangs through a simple ligation process. Next generation sequencing analysis of the assembled DNA library revealed that ligation was accurate, directional and unbiased. This straightforward DNA assembly method should fulfill the versatile needs of both in vivo and in vitro functional screening of random peptides and RNA created with a desired amino acid and nucleotide composition, as well as making highly repetitive gene constructs that are difficult to synthesize de novo. PMID:26010273

  10. Selection of ceramic fluorapatite-binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography.

    PubMed

    Islam, Tuhidul; Bibi, Noor Shad; Vennapusa, Rami Reddy; Fernandez-Lahore, Marcelo

    2013-08-01

    Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA-specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N-terminal sequence was found in two selected peptides: F4-2 (KPRSMLH) and F5-4 (KPRSVSG). The peptide F5-4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5-4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage-derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins.

  11. 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.

  12. 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/.

  13. Design, synthesis, and application of OB2C combinatorial peptide and peptidomimetic libraries.

    PubMed

    Liu, Ruiwu; Shih, Tsung-Chieh; Deng, Xiaojun; Anwar, Lara; Ahadi, Sara; Kumaresan, Pappanaicken; Lam, Kit S

    2015-01-01

    The "one-bead two-compound" (OB2C) combinatorial library is constructed on topologically segregated trifunctional bilayer beads such that each bead has a fixed cell-capturing ligand and a random library compound co-displayed on its surface and a chemical coding tag (bar code) inside the bead. An OB2C library containing thousands to millions of compounds can be synthesized and screened concurrently within a short period of time. When live cells are incubated with such OB2C libraries, every bead will be coated with a monolayer of cells. The cell membranes of the captured cells facing the bead surface are exposed to the library compounds tethered to each bead. A specific biochemical or cellular response can be detected with an appropriate reporter system. The OB2C method enables investigators to rapidly discover synthetic molecules that not only interact with cell-surface receptors but can also stimulate or inhibit downstream cell signaling. To demonstrate this powerful method, one OB2C peptide library and two OB2C peptidomimetic libraries were synthesized and screened against Molt-4 lymphoma cells to discover "death ligands." Apoptosis of the bead-bound cells was detected with immunocytochemistry using horseradish peroxidase (HRP)-conjugated anti-cleaved caspase-3 antibody and 3,3'-diaminobenzidine as a substrate. Two novel synthetic "death ligands" against Molt-4 cells were discovered using this OB2C library approach.

  14. Screening and characterization of anti-SEB peptides using a bacterial display library and microfluidic magnetic sorting

    PubMed Central

    Kogot, Joshua M; Pennington, Joseph M; Sarkes, Deborah A; Kingery, David A; Pellegrino, Paul M; Stratis-Cullum, Dimitra N

    2014-01-01

    Bacterial peptide display libraries enable the rapid and efficient selection of peptides that have high affinity and selectivity toward their targets. Using a 15-mer random library on the outer surface of Escherichia coli (E.coli), high-affinity peptides were selected against a staphylococcal enterotoxin B (SEB) protein after four rounds of biopanning. On-cell screening analysis of affinity and specificity were measured by flow cytometry and directly compared to the synthetic peptide, off-cell, using peptide-ELISA. DNA sequencing of the positive clones after four rounds of microfluidic magnetic sorting (MMS) revealed a common consensus sequence of (S/T)CH(Y/F)W for the SEB-binding peptides R338, R418, and R445. The consensus sequence in these bacterial display peptides has similar amino acid characteristics with SEB peptide sequences isolated from phage display. The Kd measured by peptide-ELISA off-cell was 2.4 nM for R418 and 3.0 nM for R445. The bacterial peptide display methodology using the semiautomated MMS resulted in the discovery of selective peptides with affinity for a food safety and defense threat. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Journal of Molecular Recognition published by John Wiley & Sons, Ltd. PMID:25319622

  15. Mixture-based combinatorial libraries from small individual peptide libraries: a case study on α1-antitrypsin deficiency.

    PubMed

    Chang, Yi-Pin; Chu, Yen-Ho

    2014-05-16

    The design, synthesis and screening of diversity-oriented peptide libraries using a "libraries from libraries" strategy for the development of inhibitors of α1-antitrypsin deficiency are described. The major buttress of the biochemical approach presented here is the use of well-established solid-phase split-and-mix method for the generation of mixture-based libraries. The combinatorial technique iterative deconvolution was employed for library screening. While molecular diversity is the general consideration of combinatorial libraries, exquisite design through systematic screening of small individual libraries is a prerequisite for effective library screening and can avoid potential problems in some cases. This review will also illustrate how large peptide libraries were designed, as well as how a conformation-sensitive assay was developed based on the mechanism of the conformational disease. Finally, the combinatorially selected peptide inhibitor capable of blocking abnormal protein aggregation will be characterized by biophysical, cellular and computational methods.

  16. Random Thoughts about Medical Library Planning *

    PubMed Central

    Fry, Alderson

    1965-01-01

    There seems to be enough material about library planning, but since mistakes, obvious ones, are made, maybe it is information without meaning. Sources of planning material are discussed, and some new libraries are considered. The necessity of discarding is urged, and methods of bringing it about are suggested. PMID:14306023

  17. 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.

  18. Dynamic Peptide Library for the Discovery of Charge Transfer Hydrogels.

    PubMed

    Berdugo, Cristina; Nalluri, Siva Krishna Mohan; Javid, Nadeem; Escuder, Beatriu; Miravet, Juan F; Ulijn, Rein V

    2015-11-25

    Coupling of peptide self-assembly to dynamic sequence exchange provides a useful approach for the discovery of self-assembling materials. In here, we demonstrate the discovery and optimization of aqueous, gel-phase nanostructures based on dynamically exchanging peptide sequences that self-select to maximize charge transfer of n-type semiconducting naphthalenediimide (NDI)-dipeptide bioconjugates with various π-electron-rich donors (dialkoxy/hydroxy/amino-naphthalene or pyrene derivatives). These gel-phase peptide libraries are characterized by spectroscopy (UV-vis and fluorescence), microscopy (TEM), HPLC, and oscillatory rheology and it is found that, of the various peptide sequences explored (tyrosine Y-NDI with tyrosine Y, phenylalanine F, leucine L, valine V, alanine A or glycine G-NH2), the optimum sequence is tyrosine-phenylalanine in each case; however, both its absolute and relative yield amplification is dictated by the properties of the donor component, indicating cooperativity of peptide sequence and donor/acceptor pairs in assembly. The methodology provides an in situ discovery tool for nanostructures that enable dynamic interfacing of supramolecular electronics with aqueous (biological) systems. PMID:26540455

  19. Peptide based diagnostics: are random-sequence peptides more useful than tiling proteome sequences?

    PubMed

    Navalkar, Krupa Arun; Johnston, Stephan Albert; Stafford, Phillip

    2015-02-01

    Diagnostics using peptide ligands have been available for decades. However, their adoption in diagnostics has been limited, not because of poor sensitivity but in many cases due to diminished specificity. Numerous reports suggest that protein-based rather than peptide-based disease detection is more specific. We examined two different approaches to peptide-based diagnostics using Coccidioides (aka Valley Fever) as the disease model. Although the pathogen was discovered more than a century ago, a highly sensitive diagnostic remains unavailable. We present a case study where two different approaches to diagnosing Valley Fever were used: first, overlapping Valley Fever epitopes representing immunodominant Coccidioides antigens were tiled using a microarray format of presynthesized peptides. Second, a set of random sequence peptides identified using a 10,000 peptide immunosignaturing microarray was compared for sensitivity and specificity. The scientific hypothesis tested was that actual epitope peptides from Coccidioides would provide sufficient sensitivity and specificity as a diagnostic. Results demonstrated that random sequence peptides exhibited higher accuracy when classifying different stages of Valley Fever infection vs. epitope peptides. The epitope peptide array did provide better performance than the existing immunodiffusion array, but when directly compared to the random sequence peptides, reported lower overall accuracy. This study suggests that there are competing aspects of antibody recognition that involve conservation of pathogen sequence and aspects of mimotope recognition and amino acid substitutions. These factors may prove critical when developing the next generation of high-performance immunodiagnostics.

  20. N-Terminal Peptide Detection with Optimized Peptide-Spectrum Matching and Streamlined Sequence Libraries.

    PubMed

    Lycette, Brynne E; Glickman, Jacob W; Roth, Samuel J; Cram, Abigail E; Kim, Tae Hee; Krizanc, Danny; Weir, Michael P

    2016-09-01

    We identified tryptic peptides in yeast cell lysates that map to translation initiation sites downstream of the annotated start sites using the peptide-spectrum matching algorithms OMSSA and Mascot. To increase the accuracy of peptide-spectrum matching, both algorithms were run using several standardized parameter sets, and Mascot was run utilizing a, b, and y ions from collision-induced dissociation. A large fraction (22%) of the detected N-terminal peptides mapped to translation initiation downstream of the annotated initiation sites. Expression of several truncated proteins from downstream initiation in the same reading frame as the full-length protein (frame 1) was verified by western analysis. To facilitate analysis of the larger proteome of Drosophila, we created a streamlined sequence library from which all duplicated trypsin fragments had been removed. OMSSA assessment using this "stripped" library revealed 171 peptides that map to downstream translation initiation sites, 76% of which are in the same reading frame as the full-length annotated proteins, although some are in different reading frames creating new protein sequences not in the annotated proteome. Sequences surrounding implicated downstream AUG start codons are associated with nucleotide preferences with a pronounced three-base periodicity N1^G2^A3.

  1. Uniqueness: skews bit occurrence frequencies in randomly generated fingerprint libraries.

    PubMed

    Chen, Nelson G

    2016-08-01

    Requiring that randomly generated chemical fingerprint libraries have unique fingerprints such that no two fingerprints are identical causes a systematic skew in bit occurrence frequencies, the proportion at which specified bits are set. Observed frequencies (O) at which each bit is set within the resulting libraries systematically differ from frequencies at which bits are set at fingerprint generation (E). Observed frequencies systematically skew toward 0.5, with the effect being more pronounced as library size approaches the compound space, which is the total number of unique possible fingerprints given the number of bit positions each fingerprint contains. The effect is quantified for varying library sizes as a fraction of the overall compound space, and for changes in the specified frequency E. The cause and implications for this systematic skew are subsequently discussed. When generating random libraries of chemical fingerprints, the imposition of a uniqueness requirement should either be avoided or taken into account.

  2. Latest Advances in OBOC Peptide Libraries. Improvements in Screening Strategies and Enlarging the Family From Linear to Cyclic Libraries.

    PubMed

    Martínez-Ceron, Maria C; Giudicessi, Silvana L; Saavedra, Soledad L; Gurevich-Messina, Juan M; Erra-Balsells, Rosa; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A

    2016-01-01

    Solid phase screenings of one bead one compound (OBOC) libraries have been widely used to find ligands with pharmacological and analytical uses, and to purify or detect proteins in complex mixtures. To improve library screening, in the last years various strategies have been developed to avoid the selection of false positive beads and to obtain selective ligands. Currently, there is great interest in cyclic peptides because of their resistance to enzymatic degradation and higher selectivity compared to their linear counterparts. Lots of cyclic peptide libraries protocols have been recently developed to facilitate hits analysis. The aim of this review is to summarize the latest applications of solid phase screening of OBOC combinatorial peptide libraries, the improvements in the screening methods including mass spectrometry MS/MS techniques and the strategies to synthesize OBOC cyclic peptide libraries.

  3. RNGAVXLIB: Program library for random number generation, AVX realization

    NASA Astrophysics Data System (ADS)

    Guskova, M. S.; Barash, L. Yu.; Shchur, L. N.

    2016-03-01

    We present the random number generator (RNG) library RNGAVXLIB, which contains fast AVX realizations of a number of modern random number generators, and also the abilities to jump ahead inside a RNG sequence and to initialize up to 1019 independent random number streams with block splitting method. Fast AVX implementations produce exactly the same output sequences as the original algorithms. Usage of AVX vectorization allows to substantially improve performance of the generators. The new realizations are up to 2 times faster than the SSE realizations implemented in the previous version of the library (Barash and Shchur, 2013), and up to 40 times faster compared to the original algorithms written in ANSI C.

  4. SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries.

    PubMed

    Wu, Jemma X; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P

    2016-07-01

    The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries.

  5. Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

    PubMed Central

    Gruber, Christian W.; Muttenthaler, Markus; Freissmuth, Michael

    2016-01-01

    G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30–50%). Closer scrutiny, however, shows that only a modest fraction of (~60) GPCRs are, in fact, exploited as drug targets, only ~20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the “orthosteric site”). These additional sites include (i) binding sites for ligands (referred to as “allosteric ligands”) that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points. PMID:20687879

  6. Error analysis of deep sequencing of phage libraries: peptides censored in sequencing.

    PubMed

    Matochko, Wadim L; Derda, Ratmir

    2013-01-01

    Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N × 1 frequency vector n = ||ni||, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N × N matrix and a stochastic sampling operator (Sa). The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of Sa and use them to define the sequencing operator (Seq). Sequencing without any bias and errors is Seq = Sa IN, where IN is a N × N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (CEN), which describes elimination or statistically significant downsampling, of specific reads during the sequencing process. PMID:24416071

  7. Strategies and computational tools for improving randomized protein libraries.

    PubMed

    Patrick, Wayne M; Firth, Andrew E

    2005-10-01

    In the last decade, directed evolution has become a routine approach for engineering proteins with novel or altered properties. Concurrently, a trend away from purely 'blind' randomization strategies and towards more 'semi-rational' approaches has also become apparent. In this review, we discuss ways in which structural information and predictive computational tools are playing an increasingly important role in guiding the design of randomized libraries: web servers such as ConSurf-HSSP and SCHEMA allow the prediction of sites to target for producing functional variants, while algorithms such as GLUE, PEDEL and DRIVeR are useful for estimating library completeness and diversity. In addition, we review recent methodological developments that facilitate the construction of unbiased libraries, which are inherently more diverse than biased libraries and therefore more likely to yield improved variants.

  8. Isolation and characterization of anti-SEB peptides using magnetic sorting and bacterial peptide display library technology

    NASA Astrophysics Data System (ADS)

    Pennington, Joseph M.; Kogot, Joshua M.; Sarkes, Deborah A.; Pellegrino, Paul M.; Stratis-Cullum, Dimitra N.

    2012-06-01

    Peptide display libraries offer an alternative method to existing antibody development methods enabling rapid isolation of highly stable reagents for detection of new and emerging biological threats. Bacterial display libraries are used to isolate new peptide reagents within 1 week, which is simpler and timelier than using competing display library technology based on phage or yeast. Using magnetic sorting methods, we have isolated peptide reagents with high affinity and specificity to staphylococcal enterotoxin B (SEB), a suspected food pathogen. Flow cytometry methods were used for on-cell characterization and the binding affinity (Kd) of this new peptide reagent was determined to be 56 nm with minimal cross-reactivity to other proteins. These results demonstrated that magnetic sorting for new reagents using bacterial display libraries is a rapid and effective method and has the potential for current and new and emerging food pathogen targets.

  9. Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library.

    PubMed

    Rhaiem, Rafik Ben; Houimel, Mehdi

    2016-07-01

    Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency

  10. CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids.

    PubMed

    Duffy, Fergal J; Verniere, Mélanie; Devocelle, Marc; Bernard, Elise; Shields, Denis C; Chubb, Anthony J

    2011-04-25

    We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie .

  11. Mimotopes of the Vi Antigen of Salmonella enterica Serovar Typhi Identified from Phage Display Peptide Library

    PubMed Central

    Tang, Swee-Seong; Tan, Wen-Siang; Devi, Shamala; Wang, Lin-Fa; Pang, Tikki; Thong, Kwai-Lin

    2003-01-01

    The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNIAHKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever. PMID:14607870

  12. Selection of peptides binding to metallic borides by screening M13 phage display libraries

    PubMed Central

    2014-01-01

    Background Metal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials. Results In this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles. Conclusions This study is, to our knowledge, the first to identify peptides that

  13. Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin

    PubMed Central

    Makartsova, Anna A.; Fomin, Alexandr S.; Nushtaeva, Anna A.; Koval, Olga A.

    2016-01-01

    A recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, induces apoptosis in cultured tumor cells. The tumor suppression efficacy of RL2 was shown against mouse hepatoma-1 cells and MDA-MB-231 human breast adenocarcinoma cells. The RL2-based therapeutic drug lactaptin is distributed evenly throughout the organism, which reduces its antitumor efficacy. In the current study, we obtained a genetic construct that allows production of the recombinant fusion protein T3-RL2, consisting of RL2 and T3 peptide (YTYDPWLIFPAN), in E. coli cells. T3 peptide was selected from a phage peptide library as a result of two screenings: in vitro using MDA-MB-231 cell culture and in vivo using a mouse xenograft model of breast cancer MDA-MB-231. It was shown that the displayed peptide T3 provides binding and internalization of phage particles by MDA-MB-231 cells and their specific accumulation in MDA-MB-231 tumor tissue. In addition, based on the nucleotide sequences coding RL2 and the known tumor-targeting peptide iRGD, we obtained genetic constructs that provide synthesis of fusion proteins RL2-iRGD and RL-iRGD-His. We studied the cytotoxic activity of fusion proteins T3-RL2, RL2-iRGD and RL-iRGD-His in vitro using MDA-MB-231 and MCF-7 human adenocarcinoma cells. The in vitro results showed that the fusion proteins inhibit proliferation of both cell cultures, and their cytotoxic activity is higher than that of RL2. In vivo experiments on the study of the antitumor efficacy of the obtained fusion proteins demonstrated that T3-RL2 protein significantly inhibits MDA-MB-231 tumor growth in a xenograft model compared with RL2, while the antitumor effect of RL2-iRGD and RL-iRGD-His proteins is comparable to the effect of RL2. PMID:27513518

  14. Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin.

    PubMed

    Nemudraya, Anna A; Makartsova, Anna A; Fomin, Alexandr S; Nushtaeva, Anna A; Koval, Olga A; Richter, Vladimir A; Kuligina, Elena V

    2016-01-01

    A recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, induces apoptosis in cultured tumor cells. The tumor suppression efficacy of RL2 was shown against mouse hepatoma-1 cells and MDA-MB-231 human breast adenocarcinoma cells. The RL2-based therapeutic drug lactaptin is distributed evenly throughout the organism, which reduces its antitumor efficacy. In the current study, we obtained a genetic construct that allows production of the recombinant fusion protein T3-RL2, consisting of RL2 and T3 peptide (YTYDPWLIFPAN), in E. coli cells. T3 peptide was selected from a phage peptide library as a result of two screenings: in vitro using MDA-MB-231 cell culture and in vivo using a mouse xenograft model of breast cancer MDA-MB-231. It was shown that the displayed peptide T3 provides binding and internalization of phage particles by MDA-MB-231 cells and their specific accumulation in MDA-MB-231 tumor tissue. In addition, based on the nucleotide sequences coding RL2 and the known tumor-targeting peptide iRGD, we obtained genetic constructs that provide synthesis of fusion proteins RL2-iRGD and RL-iRGD-His. We studied the cytotoxic activity of fusion proteins T3-RL2, RL2-iRGD and RL-iRGD-His in vitro using MDA-MB-231 and MCF-7 human adenocarcinoma cells. The in vitro results showed that the fusion proteins inhibit proliferation of both cell cultures, and their cytotoxic activity is higher than that of RL2. In vivo experiments on the study of the antitumor efficacy of the obtained fusion proteins demonstrated that T3-RL2 protein significantly inhibits MDA-MB-231 tumor growth in a xenograft model compared with RL2, while the antitumor effect of RL2-iRGD and RL-iRGD-His proteins is comparable to the effect of RL2. PMID:27513518

  15. Intracellular screening of a peptide library to derive a potent peptide inhibitor of α-synuclein aggregation.

    PubMed

    Cheruvara, Harish; Allen-Baume, Victoria L; Kad, Neil M; Mason, Jody M

    2015-03-20

    Aggregation of α-synuclein (α-syn) into toxic fibrils is a pathogenic hallmark of Parkinson disease (PD). Studies have focused largely on residues 71-82, yet most early-onset mutations are located between residues 46 and 53. A semirationally designed 209,952-member library based entirely on this region was constructed, containing all wild-type residues and changes associated with early-onset PD. Intracellular cell survival screening and growth competition isolated a 10-residue peptide antagonist that potently inhibits α-syn aggregation and associated toxicity at a 1:1 stoichiometry. This was verified using continuous growth measurements and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity studies. Atomic force microscopy and circular dichroism on the same samples showed a random-coil structure and no oligomers. A new region of α-syn for inhibitor targeting has been highlighted, together with the approach of using a semirational design and intracellular screening. The peptides can then be used as candidates for modification in drugs capable of slowing or even preventing the onset of PD.

  16. Intracellular Screening of a Peptide Library to Derive a Potent Peptide Inhibitor of α-Synuclein Aggregation*

    PubMed Central

    Cheruvara, Harish; Allen-Baume, Victoria L.; Kad, Neil M.; Mason, Jody M.

    2015-01-01

    Aggregation of α-synuclein (α-syn) into toxic fibrils is a pathogenic hallmark of Parkinson disease (PD). Studies have focused largely on residues 71–82, yet most early-onset mutations are located between residues 46 and 53. A semirationally designed 209,952-member library based entirely on this region was constructed, containing all wild-type residues and changes associated with early-onset PD. Intracellular cell survival screening and growth competition isolated a 10-residue peptide antagonist that potently inhibits α-syn aggregation and associated toxicity at a 1:1 stoichiometry. This was verified using continuous growth measurements and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity studies. Atomic force microscopy and circular dichroism on the same samples showed a random-coil structure and no oligomers. A new region of α-syn for inhibitor targeting has been highlighted, together with the approach of using a semirational design and intracellular screening. The peptides can then be used as candidates for modification in drugs capable of slowing or even preventing the onset of PD. PMID:25616660

  17. A simple and effective method for producing nonrandom peptide libraries using cotton as a carrier in continuous flow peptide synthesizers.

    PubMed

    Mutulis, Felikss; Tysk, Marcus; Mutule, Ilze; Wikberg, Jarl E S

    2003-01-01

    A method has been developed for generating nonrandom peptide libraries on cotton. Disks of cotton fabric were chemically modified to enable peptide synthesis. Incorporation of a 6-aminocaproic acid residue handle on the cellulose turned out to be advantageous. Disks were labeled with silver ink, stacked one on top of another in a continuous flow peptide synthesizer column, and simultaneously subjected to automated synthesis procedures. Depending on the sequences to be synthesized, the automatic synthesis procedure was stopped, and the disks were removed from the column, sorted, and reapplied to subsequent synthesis steps. In this way, individual peptides could be easily prepared in milligram quantities on each of the cotton disks.

  18. Expanding tandem mass spectral libraries of phosphorylated peptides: advances and applications.

    PubMed

    Hu, Yingwei; Lam, Henry

    2013-12-01

    The identification of phosphorylated proteins remains a challenge in proteomics, partially due to the difficulty in assigning tandem mass (MS/MS) spectra to their originating peptide sequences with correct phosphosite localization. Because of its advantages in efficiency and sensitivity, spectral library searching is a promising alternative to conventional sequence database searching. Our work aims to construct the largest collision-induced dissociation (CID) MS/MS spectral libraries of phosphorylated peptides in human (Homo sapiens) and four model organisms (Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus) to date, to facilitate phosphorylated peptide identification by spectral library searching. We employed state-of-the-art search methods to published data and applied two recently published phosphorylation site localization tools (PhosphoRS and PTMProphet) to ascertain the phosphorylation sites. To further increase the coverage of this library, we predicted "semi-empirical" spectra for peptides containing known phosphorylation sites from the corresponding template unphosphorylated peptide spectra. The performance of the spectral libraries built were evaluated and found to be superior to conventional database searching in terms of sensitivity. Updated spectral libraries of phosphorylated peptides are made freely available for use with the spectral search engine SpectraST. The work flow being developed will be used to continuously update the libraries when new data become available.

  19. GASPRNG: GPU accelerated scalable parallel random number generator library

    NASA Astrophysics Data System (ADS)

    Gao, Shuang; Peterson, Gregory D.

    2013-04-01

    Graphics processors represent a promising technology for accelerating computational science applications. Many computational science applications require fast and scalable random number generation with good statistical properties, so they use the Scalable Parallel Random Number Generators library (SPRNG). We present the GPU Accelerated SPRNG library (GASPRNG) to accelerate SPRNG in GPU-based high performance computing systems. GASPRNG includes code for a host CPU and CUDA code for execution on NVIDIA graphics processing units (GPUs) along with a programming interface to support various usage models for pseudorandom numbers and computational science applications executing on the CPU, GPU, or both. This paper describes the implementation approach used to produce high performance and also describes how to use the programming interface. The programming interface allows a user to be able to use GASPRNG the same way as SPRNG on traditional serial or parallel computers as well as to develop tightly coupled programs executing primarily on the GPU. We also describe how to install GASPRNG and use it. To help illustrate linking with GASPRNG, various demonstration codes are included for the different usage models. GASPRNG on a single GPU shows up to 280x speedup over SPRNG on a single CPU core and is able to scale for larger systems in the same manner as SPRNG. Because GASPRNG generates identical streams of pseudorandom numbers as SPRNG, users can be confident about the quality of GASPRNG for scalable computational science applications. Catalogue identifier: AEOI_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEOI_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing provisions: UTK license. No. of lines in distributed program, including test data, etc.: 167900 No. of bytes in distributed program, including test data, etc.: 1422058 Distribution format: tar.gz Programming language: C and CUDA. Computer: Any PC or

  20. Synthesis and screening of support-bound combinatorial peptide libraries with free C-termini: determination of the sequence specificity of PDZ domains.

    PubMed

    Joo, Sang Hoon; Pei, Dehua

    2008-03-01

    Preparation of support-bound combinatorial peptide libraries with free C-termini has been challenging in the past because solid-phase peptide synthesis usually starts from the C-terminus, which must be covalently attached to the solid support. In this work, we have developed a general methodology to synthesize and screen one-bead-one-compound peptide libraries containing free C-termini. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers, and peptides were synthesized on the beads in the conventional C --> N manner, with their C-termini attached to the support through an ester linkage on the bead surface but through an amide bond in the bead interior. The surface peptides were cyclized between their N-terminal amine and a carboxyl group installed at a C-terminal linker sequence, while the internal peptides were kept in the linear form. Base hydrolysis of the ester linkage in the cyclic peptides regenerated linear peptides that contained a free alpha-carboxyl group at their C-termini but remained covalently attached to the resin via the N-termini ("inverted" peptides). An inverted peptide library containing five random residues (theoretical diversity of 3.2 x 10 (6)) was synthesized and screened for binding to four postsynaptic density-95/discs large/zona occluden-1 (PDZ) domains of sodium-hydrogen exchanger regulatory factor-1 (NHERF1) and channel-interacting PDZ domain protein (CIPP). The identity of the binding peptides was determined by sequencing the linear encoding peptides inside the bead by partial Edman degradation/mass spectrometry. Consensus recognition motifs were identified for the PDZ domains, and representative peptides were resynthesized and confirmed for binding to their cognate PDZ domains. This method should be generally applicable to all PDZ domains as well as other protein domains and enzymes that recognize the C-terminus of their target proteins.

  1. Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: deconvolution strategy.

    PubMed

    Moore, Graham J; Moore, Diana M; Roy, Samir S; Hayden, Lawrence J; Hamilton, Murray G; Chan, Nora W C; Lee, William E

    2006-02-01

    Abstract Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X1-X2-hinge-X3-X4-NH2 (capped) and X1-hinge-X2-X3 (uncapped), where X1 through X4 are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a

  2. 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.

  3. Denoising peptide tandem mass spectra for spectral libraries: a Bayesian approach.

    PubMed

    Shao, Wenguang; Lam, Henry

    2013-07-01

    With the rapid accumulation of data from shotgun proteomics experiments, it has become feasible to build comprehensive and high-quality spectral libraries of tandem mass spectra of peptides. A spectral library condenses experimental data into a retrievable format and can be used to aid peptide identification by spectral library searching. A key step in spectral library building is spectrum denoising, which is best accomplished by merging multiple replicates of the same peptide ion into a consensus spectrum. However, this approach cannot be applied to "singleton spectra," for which only one observed spectrum is available for the peptide ion. We developed a method, based on a Bayesian classifier, for denoising peptide tandem mass spectra. The classifier accounts for relationships between peaks, and can be trained on the fly from consensus spectra and immediately applied to denoise singleton spectra, without hard-coded knowledge about peptide fragmentation. A linear regression model was also trained to predict the number of useful "signal" peaks in a spectrum, thereby obviating the need for arbitrary thresholds for peak filtering. This Bayesian approach accumulates weak evidence systematically to boost the discrimination power between signal and noise peaks, and produces readily interpretable conditional probabilities that offer valuable insights into peptide fragmentation behaviors. By cross validation, spectra denoised by this method were shown to retain more signal peaks, and have higher spectral similarities to replicates, than those filtered by intensity only.

  4. An extensive library of surrogate peptides for all human proteins.

    PubMed

    Mohammed, Yassene; Borchers, Christoph H

    2015-11-01

    Selecting the most appropriate surrogate peptides to represent a target protein is a major component of experimental design in Multiple Reaction Monitoring (MRM). Our software PeptidePicker with its v-score remains distinctive in its approach of integrating information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM that is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our "best knowledge" for selecting candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it has previously been observed. Here we present an updated approach where we have already compiled a list of all possible surrogate peptides of the human proteome. Using our stringent selection criteria, the list includes 165k suitable MRM peptides covering 17k proteins of the human reviewed proteins in UniProtKB. Compared to average of 2-4min per protein for retrieving and integrating the information, the precompiled list includes all peptides available instantly. This allows a more cohesive and faster design of a multiplexed MRM experiment and provides insights into evidence for a protein's existence. We will keep this list up-to-date as proteomics data repositories continue to grow. This article is part of a Special Issue entitled: Computational Proteomics. PMID:26232110

  5. 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

  6. Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries and alanine scanning.

    PubMed

    Kokoszka, Malgorzata E; Kay, Brian K

    2015-01-01

    One avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. Among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. With the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of 2 months. To illustrate this approach, we use a library of bacteriophage M13 particles, which display 12-mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the SH3 domain of the human Lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase Cbk1. The binding properties of the selected peptide ligands are then dissected by sequence alignment, Kunkel mutagenesis, and alanine scanning. Finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery. PMID:25616333

  7. Methods for the creation of cyclic Peptide libraries for use in lead discovery.

    PubMed

    Foster, Andrew D; Ingram, James D; Leitch, Eilidh K; Lennard, Katherine R; Osher, Eliot L; Tavassoli, Ali

    2015-06-01

    The identification of initial hits is a crucial stage in the drug discovery process. Although many projects adopt high-throughput screening of small-molecule libraries at this stage, there is significant potential for screening libraries of macromolecules created using chemical biology approaches. Not only can the production of the library be directly interfaced with a cell-based assay, but these libraries also require significantly fewer resources to generate and maintain. In this context, cyclic peptides are increasingly viewed as ideal scaffolds and have proven capability against challenging targets such as protein-protein interactions. Here we discuss a range of methods used for the creation of cyclic peptide libraries and detail examples of their successful implementation.

  8. Two-stage selection of sequences from a random phage display library delineates both core residues and permitted structural range within an epitope.

    PubMed

    Miceli, R M; DeGraaf, M E; Fischer, H D

    1994-01-01

    Libraries of random peptides can be screened to identify species which interact with antibodies or receptors. Similarly, maps of native molecular interactions can frequently be deduced by screening a limited set of peptide fragments derived from sequences within a native antigen or ligand. However, the existence of cross-reactive sequences that mimic original epitopes and the limited replaceability of amino acid residues suggest that the sequence space accessible by a receptor can be much broader. Definition of this space is of particular importance where structural information is required for peptidomimetic or drug design. We have used a two-stage selection scheme to expand the sequence space accessible by a phage display library and to define peptide epitopes of the anti-FLAG octapeptide monoclonal M2 antibody. Affinity selection of a primary library of 2 x 10(6) random decapeptides identified a non-contiguous core of three residues in the binding motif Tyr-Lys-Xaa-Xaa-Asp. A second stage library with 2 x 10(7) individual clones bearing the core motif but with the remaining flanking and internal residues re-randomized permitted access to a broader sequence space represented in a library equivalent to several orders of magnitude larger. Data here demonstrate that extended access to binding sequence space permitted by multi-stage screening of phage display libraries can reveal not only essential residues required for ligand binding, but also the ligand structural range permitted within the receptor binding pocket.

  9. Antiviral drug discovery strategy using combinatorial libraries of structurally constrained peptides.

    PubMed

    Real, Eléonore; Rain, Jean-Christophe; Battaglia, Véronique; Jallet, Corinne; Perrin, Pierre; Tordo, Noël; Chrisment, Peggy; D'Alayer, Jacques; Legrain, Pierre; Jacob, Yves

    2004-07-01

    We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i). an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii). a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii). an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.

  10. Selection of novel peptide mimics of the GD2 ganglioside from a constrained phage-displayed peptide library.

    PubMed

    Horwacik, Irena; Czaplicki, Dominik; Talarek, Katarzyna; Kowalczyk, Aleksandra; Bolesta, Elzbieta; Kozbor, Danuta; Rokita, Hanna

    2007-05-01

    Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualitative changes in expression of gangliosides observed in tumors of a neuroectodermal origin such as neuroblastoma, melanoma and astrocytoma. The presence of large amounts of GD2 ganglioside on neuroblastoma cells, as compared to normal cells, opens the possibilities to use the tumor-associated carbohydrate antigen in diagnosis and immunotherapeutic approaches. In the quest for immunogens potentially capable of eliciting anti-GD2 ganglioside immune responses, we performed affinity purification of phage-displayed peptides from the LX-8 library (12-mer containing disulphide bridge). The library was screened with the biotinylated anti-GD2 ganglioside 14G2a mAb monoclonal antibody. Our goal was to isolate and characterize peptide mimics of GD2 ganglioside. Numerous individual phage clones that bound 14G2a mAb were identified with the application of immunoblotting technique in the phage pools yielded from the pannings. The phage-borne peptides were tested for their anti-GD2 ganglioside antibody binding ability using ELISA. Among these clones five different phage-displayed peptide sequences were identified. Moreover, we showed that the secondary structure of the peptides, stabilized by the disulfide bridging between cysteine residues at positions 2 and 11, was crucial for the binding of the peptides to 14G2a mAb. In a separate set of experiments, we observed a competition of the peptides, expressed on phages as well as in their synthetic form, with the nominal antigen GD2 ganglioside expressed on IMR-32 neuroblastoma cells for binding to 14G2a mAb. Based on the obtained results we concluded that all of these 5 peptides were mimics of the GD2 ganglioside. PMID:17390090

  11. A large synthetic peptide and phosphopeptide reference library for mass spectrometry-based proteomics.

    PubMed

    Marx, Harald; Lemeer, Simone; Schliep, Jan Erik; Matheron, Lucrece; Mohammed, Shabaz; Cox, Jürgen; Mann, Matthias; Heck, Albert J R; Kuster, Bernhard

    2013-06-01

    We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search engines (Mascot and Andromeda) and fragmentation methods (beam-type collision-induced dissociation (HCD) and electron transfer dissociation (ETD)) for peptide identification. We also compared the sensitivities and accuracies of phosphorylation-site localization tools (Mascot Delta Score, PTM score and phosphoRS), and we characterized the chromatographic behavior of peptides in the library. We found that HCD identified more peptides and phosphopeptides than did ETD, that phosphopeptides generally eluted later from reversed-phase columns and were easier to identify than unmodified peptides and that current computational tools for proteomics can still be substantially improved. These peptides and spectra will facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns.

  12. Interconversion of Peptide Mass Spectral Libraries Derivatized with iTRAQ or TMT Labels.

    PubMed

    Zhang, Zheng; Yang, Xiaoyu; Mirokhin, Yuri A; Tchekhovskoi, Dmitrii V; Ji, Weihua; Markey, Sanford P; Roth, Jeri; Neta, Pedatsur; Hizal, Deniz Baycin; Bowen, Michael A; Stein, Stephen E

    2016-09-01

    Derivitization of peptides with isobaric tags such as iTRAQ and TMT is widely employed in proteomics due to their compatibility with multiplex quantitative measurements. We recently made publicly available a large peptide library derived from iTRAQ 4-plex labeled spectra. This resource has not been used for identifying peptides labeled with related tags with different masses, because values for virtually all masses of precursor and most product ions would differ for ions containing the different tags as well as containing different tag-specific peaks. We describe a method for interconverting spectra from iTRAQ 4-plex to TMT (6- and 10-plex) and to iTRAQ 8-plex. We interconvert spectra by appropriately mass shifting sequence ions and discarding derivative-specific peaks. After this "cleaning" of search spectra, we demonstrate that the converted libraries perform well in terms of peptide spectral matches. This is demonstrated by comparing results using sequence database searches as well as by comparing search effectiveness using original and converted libraries. At 1% FDR TMT labeled query spectra match 97% as many spectra against a converted iTRAQ library as compared to an original TMT library. Overall this interconversion strategy provides a practical way to extend results from one derivatization method to others that share related chemistry and do not significantly alter fragmentation profiles. PMID:27386737

  13. Interconversion of Peptide Mass Spectral Libraries Derivatized with iTRAQ or TMT Labels.

    PubMed

    Zhang, Zheng; Yang, Xiaoyu; Mirokhin, Yuri A; Tchekhovskoi, Dmitrii V; Ji, Weihua; Markey, Sanford P; Roth, Jeri; Neta, Pedatsur; Hizal, Deniz Baycin; Bowen, Michael A; Stein, Stephen E

    2016-09-01

    Derivitization of peptides with isobaric tags such as iTRAQ and TMT is widely employed in proteomics due to their compatibility with multiplex quantitative measurements. We recently made publicly available a large peptide library derived from iTRAQ 4-plex labeled spectra. This resource has not been used for identifying peptides labeled with related tags with different masses, because values for virtually all masses of precursor and most product ions would differ for ions containing the different tags as well as containing different tag-specific peaks. We describe a method for interconverting spectra from iTRAQ 4-plex to TMT (6- and 10-plex) and to iTRAQ 8-plex. We interconvert spectra by appropriately mass shifting sequence ions and discarding derivative-specific peaks. After this "cleaning" of search spectra, we demonstrate that the converted libraries perform well in terms of peptide spectral matches. This is demonstrated by comparing results using sequence database searches as well as by comparing search effectiveness using original and converted libraries. At 1% FDR TMT labeled query spectra match 97% as many spectra against a converted iTRAQ library as compared to an original TMT library. Overall this interconversion strategy provides a practical way to extend results from one derivatization method to others that share related chemistry and do not significantly alter fragmentation profiles.

  14. Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments.

    PubMed

    Chusacultanachai, Sudsanguan; Yuthavong, Yongyuth

    2004-01-01

    The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP). These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity. The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step. After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication. The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations. This strategy is suitable for generation of random mutations with low frequency in a large target DNA. Error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained. Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries. DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides. This strategy uses two chemically synthesized degenerate oligonucleotides as primers. By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to

  15. 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.

  16. 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

  17. Phage Peptide Libraries As a Source of Targeted Ligands

    PubMed Central

    Nemudraya, A. A.; Richter, V. A.; Kuligina, E. V.

    2016-01-01

    One of the dominant trends in modern pharmacology is the creation of drugs that act directly on the lesion focus and have minimal toxicity on healthy tissues and organs. This problem is particularly acute in relation to oncologic diseases. Short tissue- and organ-specific peptides capable of delivering drugs to the affected organ or tissue are considered promising targeted agents that can be used in the diagnosis and therapy of diseases, including cancer. The review discusses in detail the technology of phage display as a method for obtaining specific targeted peptide agents and offers examples of their use in diagnostic and clinical practice. PMID:27099784

  18. Autocrine-Based Selection of Drugs That Target Ion Channels from Combinatorial Venom Peptide Libraries.

    PubMed

    Zhang, Hongkai; Du, Mingjuan; Xie, Jia; Liu, Xiao; Sun, Jingying; Wang, Wei; Xin, Xiu; Possani, Lourival D; Yea, Kyungmoo; Lerner, Richard A

    2016-08-01

    Animal venoms represent a rich source of pharmacologically active peptides that interact with ion channels. However, a challenge to discovering drugs remains because of the slow pace at which venom peptides are discovered and refined. An efficient autocrine-based high-throughput selection system was developed to discover and refine venom peptides that target ion channels. The utility of this system was demonstrated by the discovery of novel Kv1.3 channel blockers from a natural venom peptide library that was formatted for autocrine-based selection. We also engineered a Kv1.3 blocker peptide (ShK) derived from sea anemone to generate a subtype-selective Kv1.3 blocker with a long half-life in vivo. PMID:27197631

  19. 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.

  20. 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.

  1. Extending the coverage of spectral libraries: a neighbor-based approach to predicting intensities of peptide fragmentation spectra.

    PubMed

    Ji, Chao; Arnold, Randy J; Sokoloski, Kevin J; Hardy, Richard W; Tang, Haixu; Radivojac, Predrag

    2013-03-01

    Searching spectral libraries in MS/MS is an important new approach to improving the quality of peptide and protein identification. The idea relies on the observation that ion intensities in an MS/MS spectrum of a given peptide are generally reproducible across experiments, and thus, matching between spectra from an experiment and the spectra of previously identified peptides stored in a spectral library can lead to better peptide identification compared to the traditional database search. However, the use of libraries is greatly limited by their coverage of peptide sequences: even for well-studied organisms a large fraction of peptides have not been previously identified. To address this issue, we propose to expand spectral libraries by predicting the MS/MS spectra of peptides based on the spectra of peptides with similar sequences. We first demonstrate that the intensity patterns of dominant fragment ions between similar peptides tend to be similar. In accordance with this observation, we develop a neighbor-based approach that first selects peptides that are likely to have spectra similar to the target peptide and then combines their spectra using a weighted K-nearest neighbor method to accurately predict fragment ion intensities corresponding to the target peptide. This approach has the potential to predict spectra for every peptide in the proteome. When rigorous quality criteria are applied, we estimate that the method increases the coverage of spectral libraries available from the National Institute of Standards and Technology by 20-60%, although the values vary with peptide length and charge state. We find that the overall best search performance is achieved when spectral libraries are supplemented by the high quality predicted spectra.

  2. Boronic acid functionalized peptidyl synthetic lectins: Combinatorial library design, peptide sequencing, and selective glycoprotein recognition

    PubMed Central

    Bicker, Kevin L.; Sun, Jing; Lavigne, John J.; Thompson, Paul R.

    2011-01-01

    Aberrant glycosylation of cell membrane and secreted glycoproteins is a hallmark of various disease states, including cancer. The natural lectins currently used in the recognition of these glycoproteins are costly, difficult to produce, and unstable towards rigorous use. Herein we describe the design and synthesis of several boronic acid functionalized peptide-based synthetic lectin (SL) libraries, as well as the optimized methodology for obtaining peptide sequences of these SLs. SL libraries were subsequently used to identify SLs with as high as 5-fold selectivity for various glycoproteins. SLs will inevitably find a role in cancer diagnositics, given that they do not suffer from the drawbacks of natural lectins and that the combinatorial nature of these libraries allows for the identification of an SL for nearly any glycosylated biomolecule. PMID:21405093

  3. Tandem mass spectral libraries of peptides in digests of individual proteins: Human Serum Albumin (HSA).

    PubMed

    Dong, Qian; Yan, Xinjian; Kilpatrick, Lisa E; Liang, Yuxue; Mirokhin, Yuri A; Roth, Jeri S; Rudnick, Paul A; Stein, Stephen E

    2014-09-01

    This work presents a method for creating a mass spectral library containing tandem spectra of identifiable peptide ions in the tryptic digestion of a single protein. Human serum albumin (HSA(1)) was selected for this purpose owing to its ubiquity, high level of characterization and availability of digest data. The underlying experimental data consisted of ∼3000 one-dimensional LC-ESI-MS/MS runs with ion-trap fragmentation. In order to generate a wide range of peptides, studies covered a broad set of instrument and digestion conditions using multiple sources of HSA and trypsin. Computer methods were developed to enable the reliable identification and reference spectrum extraction of all peptide ions identifiable by current sequence search methods. This process made use of both MS2 (tandem) spectra and MS1 (electrospray) data. Identified spectra were generated for 2918 different peptide ions, using a variety of manually-validated filters to ensure spectrum quality and identification reliability. The resulting library was composed of 10% conventional tryptic and 29% semitryptic peptide ions, along with 42% tryptic peptide ions with known or unknown modifications, which included both analytical artifacts and post-translational modifications (PTMs) present in the original HSA. The remaining 19% contained unexpected missed-cleavages or were under/over alkylated. The methods described can be extended to create equivalent spectral libraries for any target protein. Such libraries have a number of applications in addition to their known advantages of speed and sensitivity, including the ready re-identification of known PTMs, rejection of artifact spectra and a means of assessing sample and digestion quality.

  4. Development of a dedicated peptide tandem mass spectral library for conservation science.

    PubMed

    Fremout, Wim; Dhaenens, Maarten; Saverwyns, Steven; Sanyova, Jana; Vandenabeele, Peter; Deforce, Dieter; Moens, Luc

    2012-05-30

    In recent years, the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) on tryptic digests of cultural heritage objects has attracted much attention. It allows for unambiguous identification of peptides and proteins, and even in complex mixtures species-specific identification becomes feasible with minimal sample consumption. Determination of the peptides is commonly based on theoretical cleavage of known protein sequences and on comparison of the expected peptide fragments with those found in the MS/MS spectra. In this approach, complex computer programs, such as Mascot, perform well identifying known proteins, but fail when protein sequences are unknown or incomplete. Often, when trying to distinguish evolutionarily well preserved collagens of different species, Mascot lacks the required specificity. Complementary and often more accurate information on the proteins can be obtained using a reference library of MS/MS spectra of species-specific peptides. Therefore, a library dedicated to various sources of proteins in works of art was set up, with an initial focus on collagen rich materials. This paper discusses the construction and the advantages of this spectral library for conservation science, and its application on a number of samples from historical works of art.

  5. Stable-isotope-labeled Histone Peptide Library for Histone Post-translational Modification and Variant Quantification by Mass Spectrometry *

    PubMed Central

    Lin, Shu; Wein, Samuel; Gonzales-Cope, Michelle; Otte, Gabriel L.; Yuan, Zuo-Fei; Afjehi-Sadat, Leila; Maile, Tobias; Berger, Shelley L.; Rush, John; Lill, Jennie R.; Arnott, David; Garcia, Benjamin A.

    2014-01-01

    To facilitate accurate histone variant and post-translational modification (PTM) quantification via mass spectrometry, we present a library of 93 synthetic peptides using Protein-Aqua™ technology. The library contains 55 peptides representing different modified forms from histone H3 peptides, 23 peptides representing H4 peptides, 5 peptides representing canonical H2A peptides, 8 peptides representing H2A.Z peptides, and peptides for both macroH2A and H2A.X. The PTMs on these peptides include lysine mono- (me1), di- (me2), and tri-methylation (me3); lysine acetylation; arginine me1; serine/threonine phosphorylation; and N-terminal acetylation. The library was subjected to chemical derivatization with propionic anhydride, a widely employed protocol for histone peptide quantification. Subsequently, the detection efficiencies were quantified using mass spectrometry extracted ion chromatograms. The library yields a wide spectrum of detection efficiencies, with more than 1700-fold difference between the peptides with the lowest and highest efficiencies. In this paper, we describe the impact of different modifications on peptide detection efficiencies and provide a resource to correct for detection biases among the 93 histone peptides. In brief, there is no correlation between detection efficiency and molecular weight, hydrophobicity, basicity, or modification type. The same types of modifications may have very different effects on detection efficiencies depending on their positions within a peptide. We also observed antagonistic effects between modifications. In a study of mouse trophoblast stem cells, we utilized the detection efficiencies of the peptide library to correct for histone PTM/variant quantification. For most histone peptides examined, the corrected data did not change the biological conclusions but did alter the relative abundance of these peptides. For a low-abundant histone H2A variant, macroH2A, the corrected data led to a different conclusion than the

  6. Correlated matrix-assisted laser desorption/ionization mass spectrometry and fluorescent imaging of photocleavable peptide-coded random bead-arrays

    PubMed Central

    Lim, Mark J; Liu, Ziying; Braunschweiger, Karen I; Awad, Amany; Rothschild, Kenneth J

    2013-01-01

    RATIONALE Rapidly performing global proteomic screens is an important goal in the post-genomic era. Correlated matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and fluorescent imaging of photocleavable peptide-coded random bead-arrays was evaluated as a critical step in a new method for proteomic screening that combines many of the advantages of MS with fluorescence-based microarrays. METHODS Small peptide-coded model bead libraries containing up to 20 different bead species were constructed by attaching peptides to 30–34 µm diameter glass, agarose or TentaGel® beads using photocleavable biotin or a custom-designed photocleavable linker. The peptide-coded bead libraries were randomly arrayed into custom gold-coated micro-well plates with 45 µm diameter wells and subjected to fluorescence and MALDI mass spectrometric imaging (MALDI-MSI). RESULTS Photocleavable mass-tags from individual beads in these libraries were spatially localized as ∼65 µm spots using MALDI-MSI with high sensitivity and mass resolution. Fluorescently tagged beads were identified and correlated with their matching photocleavable mass-tags by comparing the fluorescence and MALDI-MS images of the same bead-array. Post-translational modification of the peptide Kemptide was also detected on individual beads in a photocleavable peptide-coded bead-array by MALDI-MSI alone, after exposure of the beads to protein kinase A (PKA). CONCLUSIONS Correlated MALDI-MS and fluorescent imaging of photocleavable peptide-coded random bead-arrays can provide a basis for performing global proteomic screening. © 2013 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons, Ltd. PMID:24285390

  7. Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries.

    SciTech Connect

    Kay, B. K.; Castagnoli, L.; Biosciences Division; Univ. of Rome

    2003-01-01

    This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

  8. Effect of peptide assay library size and composition in targeted data-independent acquisition-MS analyses.

    PubMed

    Parker, Sarah J; Venkatraman, Vidya; Van Eyk, Jennifer E

    2016-08-01

    The quantification of peptides using targeted analysis of data-independent acquisition MS (DIA-MS) is dependent on the size and characteristics of the assay library. We addressed several important questions on how library composition influences: (1) the number of peptides extracted from DIA-MS datasets, (2) the quality of these peptides and proteins, and (3) the biological conclusions inferred. To answer these questions we constructed five libraries from mouse vascular smooth muscle cell (VSMC) lysate, each unique in depth, input sample complexity, data acquisition mode (DDA-MS or DIA-MS), and precursor fragmentation mode (TOF-CID or Orbitrap HCD) and extracted them against the same eight DIA-MS files of VSMCs treated with vehicle or transforming growth factor β-1 (TGF-β1). We found that along with differences in peptide and protein composition, the fragments representing a given peptide differed between the libraries. Collectively these differences impacted both peak group score profile and protein abundance estimates. Surprisingly, there was little overlap in the TGF-β1 response proteome between libraries. We conclude that additional work is needed to optimize peptide assay library building for DIA-MS applications, particularly in terms of selecting optimal peptides and their respective fragments for protein quantification. PMID:27432805

  9. Panning of a phage display library against a synthetic capsule for peptide ligands that bind to the native capsule of Bacillus anthracis.

    PubMed

    Beer, Michael; Liu, Chun-Qiang

    2012-01-01

    Bacillus anthracis is the causative agent of anthrax with the ability to not only produce a tripartite toxin, but also an enveloping capsule comprised primarily of γ-D-glutamic acid residues. The purpose of this study was to isolate peptide ligands capable of binding to the native capsule of B. anthracis from a commercial phage display peptide library using a synthetic form of the capsule consisting of 12 γ-D-glutamic acid residues. Following four rounds of selection, 80 clones were selected randomly and analysed by DNA sequencing. Four clones, each containing a unique consensus sequence, were identified by sequence alignment analysis. Phage particles were prepared and their derived 12-mer peptides were also chemically synthesized and conjugated to BSA. Both the phage particles and free peptide-BSA conjugates were evaluated by ELISA for binding to encapsulated cells of B. anthracis as well as a B. anthracis capsule extract. All the phage particles tested except one were able to bind to both the encapsulated cells and the capsule extract. However, the peptide-BSA conjugates could only bind to the encapsulated cells. One of the peptide-BSA conjugates, with the sequence DSSRIPMQWHPQ (termed G1), was fluorescently labelled and its binding to the encapsulated cells was further confirmed by confocal microscopy. The results demonstrated that the synthetic capsule was effective in isolating phage-displayed peptides with binding affinity for the native capsule of B. anthracis.

  10. 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

  11. 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.

  12. Molecular diversification in spider venoms: a web of combinatorial peptide libraries.

    PubMed

    Escoubas, Pierre

    2006-11-01

    Spider venoms are a rich source of novel pharmacologically and agrochemically interesting compounds that have received increased attention from pharmacologists and biochemists in recent years. The application of technologies derived from genomics and proteomics have led to the discovery of the enormous molecular diversity of those venoms, which consist mainly of peptides and proteins. The molecular diversity of spider peptides has been revealed by mass spectrometry and appears to be based on a limited set of structural scaffolds. Genetic analysis has led to a further understanding of the molecular evolution mechanisms presiding over the generation of these combinatorial peptide libraries. Gene duplication and focal hypermutation, which has been described in cone snails, appear to be common mechanisms to venomous mollusks and spiders. Post-translational modifications, fine structural variations and new molecular scaffolds are other potential mechanisms of toxin diversification, leading to the pharmacologically complex cocktails used for predation and defense.

  13. Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection.

    PubMed

    Wang, Yongping; Speier, Jacqueline S; Engram-Pearl, Jessica; Wilson, Robert B

    2014-01-01

    RNA interference (RNAi) is a mechanism for interfering with gene expression through the action of small, non-coding RNAs. We previously constructed a short-hairpin-loop RNA (shRNA) encoding library that is random at the nucleotide level [1]. In this library, the stems of the hairpin are completely complementary. To improve the potency of initial hits, and therefore signal-to-noise ratios in library screening, as well as to simplify hit-sequence retrieval by PCR, we constructed a second-generation library in which we introduced random mismatches between the two halves of the stem of each hairpin, on a random template background. In a screen for shRNAs that protect an interleukin-3 (IL3) dependent cell line from IL3 withdrawal, our second-generation library yielded hit sequences with significantly higher potencies than those from the first-generation library in the same screen. Our method of random mutagenesis was effective for a random template and is likely suitable, therefore, for any DNA template of interest. The improved potency of our second-generation library expands the range of possible unbiased screens for small-RNA therapeutics and biologic tools.

  14. A polystyrene binding target-unrelated peptide isolated in the screening of phage display library.

    PubMed

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2016-11-01

    Phage display is a powerful methodology for the identification of peptide ligands binding to any desired target. However, the selection of target-unrelated peptides (TUPs) appears as a huge problem in the screening of phage display libraries through biopanning. The phage-displayed peptide TLHPAAD has been isolated both in our laboratory and by another reserach group on completely different screening targets prompting us to hypothesize that it may be a potential TUP. In the current study, we analyzed the binding characteristics and propagation rate of phage clone displaying TLHPAAD peptide (SW-TUP clone). The results of ELISA experiment and phage recovery assay provided strong support for the notion that SW-TUP phage binds to polystyrene with a significantly higher affinity than control phage clones. Furthermore, this polystyrene binding was demonstrated to occur in a concentration- and pH-dependent mode. Characterization of the propagation profile of phage clones within a specified time course revealed no statistically significant difference between the amplification rate of SW-TUP and control phages. Our findings lead us to the conclusion that SW-TUP phage clone with the displayed peptide TLHPAAD is not a true target binder and its selection in biopanning experiments results from its bidning affinity to the polystyrene surface of the solid phase.

  15. A polystyrene binding target-unrelated peptide isolated in the screening of phage display library.

    PubMed

    Bakhshinejad, Babak; Sadeghizadeh, Majid

    2016-11-01

    Phage display is a powerful methodology for the identification of peptide ligands binding to any desired target. However, the selection of target-unrelated peptides (TUPs) appears as a huge problem in the screening of phage display libraries through biopanning. The phage-displayed peptide TLHPAAD has been isolated both in our laboratory and by another reserach group on completely different screening targets prompting us to hypothesize that it may be a potential TUP. In the current study, we analyzed the binding characteristics and propagation rate of phage clone displaying TLHPAAD peptide (SW-TUP clone). The results of ELISA experiment and phage recovery assay provided strong support for the notion that SW-TUP phage binds to polystyrene with a significantly higher affinity than control phage clones. Furthermore, this polystyrene binding was demonstrated to occur in a concentration- and pH-dependent mode. Characterization of the propagation profile of phage clones within a specified time course revealed no statistically significant difference between the amplification rate of SW-TUP and control phages. Our findings lead us to the conclusion that SW-TUP phage clone with the displayed peptide TLHPAAD is not a true target binder and its selection in biopanning experiments results from its bidning affinity to the polystyrene surface of the solid phase. PMID:27555439

  16. GLUE-IT and PEDEL-AA: new programmes for analyzing protein diversity in randomized libraries.

    PubMed

    Firth, Andrew E; Patrick, Wayne M

    2008-07-01

    There are many methods for introducing random mutations into nucleic acid sequences. Previously, we described a suite of programmes for estimating the completeness and diversity of randomized DNA libraries generated by a number of these protocols. Our programmes suggested some empirical guidelines for library design; however, no information was provided regarding library diversity at the protein (rather than DNA) level. We have now updated our web server, enabling analysis of translated libraries constructed by site-saturation mutagenesis and error-prone PCR (epPCR). We introduce GLUE-Including Translation (GLUE-IT), which finds the expected amino acid completeness of libraries in which up to six codons have been independently varied (according to any user-specified randomization scheme). We provide two tools for assisting with experimental design: CodonCalculator, for assessing amino acids corresponding to given randomized codons; and AA-Calculator, for finding degenerate codons that encode user-specified sets of amino acids. We also present PEDEL-AA, which calculates amino acid statistics for libraries generated by epPCR. Input includes the parent sequence, overall mutation rate, library size, indel rates and a nucleotide mutation matrix. Output includes amino acid completeness and diversity statistics, and the number and length distribution of sequences truncated by premature termination codons. The web interfaces are available at http://guinevere.otago.ac.nz/stats.html.

  17. Blocking peptides against HBV: PreS1 protein selected from a phage display library

    SciTech Connect

    Wang, Wei; Liu, Yang; Zu, Xiangyang; Jin, Rui; Xiao, Gengfu

    2011-09-09

    Highlights: {yields} Successfully selected specific PreS1-interacting peptides by using phage displayed library. {yields} Alignment of the positive phage clones revealed a consensus PreS1 binding motif. {yields} A highly enriched peptide named P7 had a strong binding ability for PreS1. {yields} P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX{sub n}HX{sub m}HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.

  18. SUPERFICIAL – Surface mapping of proteins via structure-based peptide library design

    PubMed Central

    Goede, Andrean; Jaeger, Ines S; Preissner, Robert

    2005-01-01

    Background The determination of protein surfaces and the detection of binding sites are essential to our understanding of protein-protein interactions. Such binding sites can be characterised as linear and non-linear, the non-linear sites being prevailant. Conventional mapping techniques with arrays of synthetic peptides have limitations with regard to the location of discontinuous or non-linear binding sites of proteins. Results We present a structure-based approach to the design of peptide libraries that mimic the whole surface or a particular region of a protein. Neighbouring sequence segments are linked by short spacers to conserve local conformation. To this end, we have developed SUPERFICIAL, a program that uses protein structures as input and generates library proposals consisting of linear and non-linear peptides. This process can be influenced by a graphical user interface at different stages, from the surface computation up to the definition of spatial regions. Conclusion Based on 3D structures, SUPERFICIAL may help to negotiate some of the existing limitations, since binding sites consisting of several linear pieces can now be detected. PMID:16153304

  19. Defining SH2 domain and PTP specificity by screening combinatorial peptide libraries

    PubMed Central

    Wavreille, Anne-Sophie; Garaud, Mathieu; Zhang, Yanyan; Pei, Dehua

    2007-01-01

    Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing short phosphotyrosyl (pY) peptide motifs in their partner proteins. Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of pY proteins, counteracting the protein tyrosine kinases. Both types of proteins exhibit primary sequence specificity, which plays at least a partial role in dictating their physiological interacting partners or substrates. A combinatorial peptide library method has been developed to systematically assess the sequence specificity of SH2 domains and PTPs. A “one-bead-one-compound” pY peptide library is synthesized on 90-μm TenteGel beads and screened against an SH2 domain or PTP of interest for binding or catalysis. The beads that carry the tightest binding sequences against the SH2 domain or the most efficient substrates of the PTP are selected by an enzyme-linked assay and individually sequenced by a partial Edman degradation/mass spectrometry technique. The combinatorial method has been applied to determine the sequence specificity of 8 SH2 domains from Src and Csk kinases, adaptor protein Grb2, and phosphatases SHP-1, SHP-2, and SHIP1 and a prototypical PTP, PTP1B. PMID:17532507

  20. 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

  1. Restriction enzyme-free construction of random gene mutagenesis libraries in Escherichia coli.

    PubMed

    Pai, Jen C; Entzminger, Kevin C; Maynard, Jennifer A

    2012-02-15

    Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in Escherichia coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restriction enzyme-free approach to library generation using megaprimers termed MegAnneal. Target DNA is first exponentially amplified using error-prone polymerase chain reaction (PCR) and then linearly amplified with a single 3' primer to generate long, randomly mutated, single-stranded megaprimers. These are annealed to single-stranded dUTP-containing template plasmid and extended with T7 polymerase to create a complementary strand, and the resulting termini are ligated with T4 DNA ligase. Using this approach, we are able to reliably generate libraries of approximately 10⁷ colony-forming units (cfu)/μg DNA/transformation in a single day. We have created MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation, and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared with current methods.

  2. Screening of integrin-binding peptides in a laminin peptide library derived from the mouse laminin β chain short arm regions.

    PubMed

    Katagiri, Fumihiko; Takagi, Masaharu; Nakamura, Minako; Tanaka, Yoichiro; Hozumi, Kentaro; Kikkawa, Yamato; Nomizu, Motoyoshi

    2014-05-15

    Laminins, major components of basement membrane, consist of three different subunits, α, β, and γ chains, and so far, five α, three β, and three γ chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin α chain sequences (α1-α5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the β1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin β2 and β3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin β1 chain, 255-266), B67 (IPYSMEYEILIRY, mouse laminin β1 chain, 604-616), B2-105 (APNFWNFTSGRG, mouse laminin β2 chain, 1081-1092), and B3-19 (GHLTGGKVQLNL, mouse laminin β3 chain, 182-193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2-105, and B3-19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with α3, α6, and β1 integrins, and B34 and B3-19 interact with β1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2-64 (LPRAMDYDLLLRW, mouse laminin β2 chain, 618-630), the Glu(8) residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide

  3. 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

  4. 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.

  5. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones.

    PubMed

    Theaker, Sarah M; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J; Cole, David K; Peakman, Mark; Sewell, Andrew K; Dolton, Garry

    2016-03-01

    Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8(+) or CD4(+) polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein-Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer.

  6. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones.

    PubMed

    Theaker, Sarah M; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J; Cole, David K; Peakman, Mark; Sewell, Andrew K; Dolton, Garry

    2016-03-01

    Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8(+) or CD4(+) polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein-Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. PMID:26826277

  7. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

    PubMed Central

    Theaker, Sarah M.; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J.; Cole, David K.; Peakman, Mark; Sewell, Andrew K.; Dolton, Garry

    2016-01-01

    Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. PMID:26826277

  8. In-depth proteomic analysis of banana (Musa spp.) fruit with combinatorial peptide ligand libraries.

    PubMed

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

    2013-01-01

    Musa ssp. is among the world's leading fruit crops. Although a strong interest on banana biochemistry exists in the scientific community, focused on metabolite composition, proteins have been scarcely investigated even if they play an important role in food allergy and stability, are a source of biologically active peptides, and can provide information about nutritional aspects of this fruit. In this work we have employed the combinatorial peptide ligand libraries after different types of protein extractions, for searching the very low-abundance proteins in banana. The use of advanced MS techniques and Musa ssp. mRNAs database in combination with the Uniprot_viridiplantae database allowed us to identify 1131 proteins. Among this huge amount of proteins we found several already known allergens such as Mus a 1, pectinesterase, superoxide dismutase, and potentially new allergens. Additionally several enzymes involved in degradation of starch granules and strictly correlated to ripening stage were identified. This is the first in-depth exploration of the banana fruit proteome and one of the largest descriptions of the proteome of any vegetable system. PMID:23161558

  9. In-depth proteomic analysis of banana (Musa spp.) fruit with combinatorial peptide ligand libraries.

    PubMed

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

    2013-01-01

    Musa ssp. is among the world's leading fruit crops. Although a strong interest on banana biochemistry exists in the scientific community, focused on metabolite composition, proteins have been scarcely investigated even if they play an important role in food allergy and stability, are a source of biologically active peptides, and can provide information about nutritional aspects of this fruit. In this work we have employed the combinatorial peptide ligand libraries after different types of protein extractions, for searching the very low-abundance proteins in banana. The use of advanced MS techniques and Musa ssp. mRNAs database in combination with the Uniprot_viridiplantae database allowed us to identify 1131 proteins. Among this huge amount of proteins we found several already known allergens such as Mus a 1, pectinesterase, superoxide dismutase, and potentially new allergens. Additionally several enzymes involved in degradation of starch granules and strictly correlated to ripening stage were identified. This is the first in-depth exploration of the banana fruit proteome and one of the largest descriptions of the proteome of any vegetable system.

  10. Chicken egg yolk cytoplasmic proteome, mined via combinatorial peptide ligand libraries.

    PubMed

    Farinazzo, Alessia; Restuccia, Umberto; Bachi, Angela; Guerrier, Luc; Fortis, Frederic; Boschetti, Egisto; Fasoli, Elisa; Citterio, Attilio; Righetti, Pier Giorgio

    2009-02-20

    The use of combinatorial peptide ligand libraries (CPLLs), containing hexapeptides terminating with a primary amine, or modified with a terminal carboxyl group, or with a terminal tertiary amine, allowed discovering and identifying a large number of previously unreported egg yolk proteins. Whereas the most comprehensive list up to date [K. Mann, M. Mann, Proteomics, 8 (2008) 178-191] tabulated about 115 unique gene products in the yolk plasma, our findings have more than doubled this value to 255 unique protein species. From the initial non-treated egg yolk it was possible to find 49 protein species; the difference was generated thanks to the use of the three combined CPLLs. The aberrant behaviour of some proteins, upon treatment via the CPLL method, such as proteins that do not interact with the library, is discussed and evaluated. Simplified elution protocols from the CPLL beads are taken into consideration, of which direct elution in a single step via sodium dodecyl sulphate desorption seems to be quite promising. Alternative methods are suggested. The list of egg yolk components here reported is by far the most comprehensive at present and could serve as a starting point for isolation and functional characterization of proteins possibly having novel pharmaceutical and biomedical applications.

  11. Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae.

    PubMed

    Rebollar, Aarón; Marcos, Jose F; López-García, Belén

    2014-11-01

    The rice blast disease caused by Magnaporthe oryzae is one of the most devastating diseases of cultivated rice. One of the most important stages in the infective cycle of M. oryzae is the formation of the dome-shaped structure called appressorium. The purpose of the present study was to identify novel peptides to control the rice blast disease by blocking the appressorium formation through screening of a synthetic peptide combinatorial library. As result of the screening, a set of 29 putative bioactive peptides were identified, synthesized and assayed in comparison with the previously identified peptide PAF104. The peptides MgAPI24, MgAPI40 and MgAPI47 showed improved inhibitory activity on the M. oryzae appressorium formation. Our data show that these peptides have a differential effect on two developmental structures: appressoria and appressorium-like structures. Antimicrobial assays against M. oryzae and other non-target microorganisms showed a weak or no toxicity of these peptides, demonstrating their specific activity blocking the appressorium formation. Therefore, the outcome of this research would be useful in the development of novel target-oriented peptides to use in plant protection.

  12. Construction, exploitation and evolution of a new peptide library displayed at high density by fusion to the major coat protein of filamentous phage.

    PubMed

    Iannolo, G; Minenkova, O; Gonfloni, S; Castagnoli, L; Cesareni, G

    1997-06-01

    The amino-terminus of the major coat protein (PVIII) of filamentous phage can be extended, up to 6-7 residues, without interfering with the phage life cycle. We have constructed a library of approximately ten millions different phage each displaying a different octapeptide joined to the amino-terminus of the 2700 copies of PVIII. Most of the resulting clones are able to produce infective particles. This molecular repertoire constituted by the periodic regular decoration of the phage filament surface, can be utilized to search elements that bind proteins or relatively small organic molecules like the textile dye Cibacron blue. By sequential growth cycles we have performed a library evolution experiment to select phage clones that have a growth advantage in the absence of any requirement for binding a specific target. The consensus of the best growers reveals a Pro rich sequence with large hydrophobic residues at position 7 and Asn at position 1 of the random peptide insert. We propose that the assembly secretion process is favoured in phages displaying this family of peptides since they fit the groove between two adjacent PVIII subunits by making advantageous molecular contacts on the phage surface. PMID:9224932

  13. Potential of Peptides as Inhibitors and Mimotopes: Selection of Carbohydrate-Mimetic Peptides from Phage Display Libraries

    PubMed Central

    Matsubara, Teruhiko

    2012-01-01

    Glycoconjugates play various roles in biological processes. In particular, oligosaccharides on the surface of animal cells are involved in virus infection and cell-cell communication. Inhibitors of carbohydrate-protein interactions are potential antiviral drugs. Several anti-influenza drugs such as oseltamivir and zanamivir are derivatives of sialic acid, which inhibits neuraminidase. However, it is very difficult to prepare a diverse range of sugar derivatives by chemical synthesis or by the isolation of natural products. In addition, the pathogenic capsular polysaccharides of bacteria are carbohydrate antigens, for which a safe and efficacious method of vaccination is required. Phage-display technology has been improved to enable the identification of peptides that bind to carbohydrate-binding proteins, such as lectins and antibodies, from a large repertoire of peptide sequences. These peptides are known as “carbohydrate-mimetic peptides (CMPs)” because they mimic carbohydrate structures. Compared to carbohydrate derivatives, it is easy to prepare mono- and multivalent peptides and then to modify them to create various derivatives. Such mimetic peptides are available as peptide inhibitors of carbohydrate-protein interactions and peptide mimotopes that are conjugated with adjuvant for vaccination. PMID:23094142

  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. PMID:23019098

  15. 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.

  16. Predicting most probable conformations of a given peptide sequence in the random coil state.

    PubMed

    Bayrak, Cigdem Sevim; Erman, Burak

    2012-11-01

    In this work, we present a computational scheme for finding high probability conformations of peptides. The scheme calculates the probability of a given conformation of the given peptide sequence using the probability distribution of torsion states. Dependence of the states of a residue on the states of its first neighbors along the chain is considered. Prior probabilities of torsion states are obtained from a coil library. Posterior probabilities are calculated by the matrix multiplication Rotational Isomeric States Model of polymer theory. The conformation of a peptide with highest probability is determined by using a hidden Markov model Viterbi algorithm. First, the probability distribution of the torsion states of the residues is obtained. Using the highest probability torsion state, one can generate, step by step, states with lower probabilities. To validate the method, the highest probability state of residues in a given sequence is calculated and compared with probabilities obtained from the Coil Databank. Predictions based on the method are 32% better than predictions based on the most probable states of residues. The ensemble of "n" high probability conformations of a given protein is also determined using the Viterbi algorithm with multistep backtracking. PMID:22955874

  17. Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

    DOEpatents

    Studier, F.W.

    1995-04-18

    Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient. 2 figs.

  18. Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

    DOEpatents

    Studier, F. William

    1995-04-18

    Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient.

  19. Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs.

    PubMed

    Gayatri, Sitaram; Cowles, Martis W; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T

    2016-01-01

    Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes - PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

  20. Widening and diversifying the proteome capture by combinatorial peptide ligand libraries via Alcian Blue dye binding.

    PubMed

    Candiano, Giovanni; Santucci, Laura; Petretto, Andrea; Lavarello, Chiara; Inglese, Elvira; Bruschi, Maurizio; Ghiggeri, Gian Marco; Boschetti, Egisto; Righetti, Pier Giorgio

    2015-01-01

    Combinatorial peptide ligand libraries (CPLLs) tend to bind complex molecules such as dyes due to their aromatic, heterocyclic, hydrophobic, and ionic nature that may affect the protein capture specificity. In this experimental work Alcian Blue 8GX, a positively charged phthalocyanine dye well-known to bind to glycoproteins and to glucosaminoglycans, was adsorbed on a chemically modified CPLL solid phase, and the behavior of the resulting conjugate was then investigated. The control and dye-adsorbed beads were used to harvest the human urinary proteome at physiological pH, this resulting in a grand total of 1151 gene products identified after the capture. Although the Alcian Blue-modified CPLL incremented the total protein capture by 115 species, it particularly enriched some families among the harvested proteins, such as glycoproteins and nucleotide-binding proteins. This study teaches that it is possible, via the two combined harvest mechanisms, to drive the CPLL capture toward the enrichment of specific protein categories. PMID:25856057

  1. Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs

    PubMed Central

    Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.

    2016-01-01

    Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

  2. A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.

    PubMed

    Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

    2015-01-01

    A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis.

  3. A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules

    PubMed Central

    Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

    2015-01-01

    A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis. PMID:26355961

  4. Advancement and applications of peptide phage display technology in biomedical science.

    PubMed

    Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung

    2016-01-01

    Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

  5. Folded Proteins Occur Frequently in Libraries of Random Amino Acid Sequences

    NASA Astrophysics Data System (ADS)

    Davidson, Alan R.; Sauer, Robert T.

    1994-03-01

    A library of synthetic genes encoding 80- to 100-residue proteins composed mainly of random combinations of glutamine (Q), leucine (L), and arginine (R) has been expressed in Escherichia coli. These genes also encode an epitope tag and six carboxyl-terminal histidines. Screening of this library by immunoblotting showed that 5% of these QLR proteins are expressed at readily detectable levels. Three well-expressed QLR proteins were purified and characterized. Each of these proteins has significant α-helical content, is largely resistant to degradation by Pronase, and has a distinct oligomeric structure. In addition, one protein unfolds in a highly cooperative manner. These properties of the QLR proteins demonstrate that they possess folded structures with some native-like properties. The QLR proteins differ from most natural proteins, however, in being remarkably resistant to denaturant-induced and thermal-induced unfolding and in being relatively insoluble in the absence of denaturants.

  6. A specific library of randomly integrated reporter genes for the isolation of inducible functions by cell sorting

    SciTech Connect

    Lapeyre, J.N.; Marini, F.; Gratzner, H.G. AMC ImmunoDiagnostics, Houston, TX )

    1993-01-01

    A library of cells containing randomly integrated reporter genes has been constructed. The purpose of this library is to enable the isolation of genes of interest which are inducible by radiation, biological response modifiers, cytokines, or other agents. These genes are located near reporter genes which can be induced by the upstream promoter of the gene of interest. The reporter gene, Lac Z, was randomly inserted into the genome by retroviral transduction and subsequent selection of the neo[sup r] gene with gentamycin. Studies of radiation inducible genes were undertaken, whereby cells with the radiation sensitive function were isolated by sorting the cells fluorescent after staining with the beta gal substrate, fluorescein digalactoside (FDG). This gene-tagging approach is an improvement over the cDNA library subtraction protocol in that a single library of cells with random marker gene integration can be repeatedly and sequentially probed by sorting under different, selective conditions, dependent upon the genes to be characterized.

  7. Selection of a potential diagnostic biomarker for HIV infection from a random library of non-biological synthetic peptoid oligomers.

    PubMed

    Gearhart, Tricia L; Montelaro, Ronald C; Schurdak, Mark E; Pilcher, Chris D; Rinaldo, Charles R; Kodadek, Thomas; Park, Yongseok; Islam, Kazi; Yurko, Raymond; Marques, Ernesto T A; Burke, Donald S

    2016-08-01

    Non-biological synthetic oligomers can serve as ligands for antibodies. We hypothesized that a random combinatorial library of synthetic poly-N-substituted glycine oligomers, or peptoids, could represent a random "shape library" in antigen space, and that some of these peptoids would be recognized by the antigen-binding pocket of disease-specific antibodies. We synthesized and screened a one bead one compound combinatorial library of peptoids, in which each bead displayed an 8-mer peptoid with ten possible different amines at each position (10(8) theoretical variants). By screening one million peptoid/beads we found 112 (approximately 1 in 10,000) that preferentially bound immunoglobulins from human sera known to be positive for anti-HIV antibodies. Reactive peptoids were then re-synthesized and rigorously evaluated in plate-based ELISAs. Four peptoids showed very good, and one showed excellent, properties for establishing a sero-diagnosis of HIV. These results demonstrate the feasibility of constructing sero-diagnostic assays for infectious diseases from libraries of random molecular shapes. In this study we sought a proof-of-principle that we could identify a potential diagnostic antibody ligand biomarker for an infectious disease in a random combinatorial library of 100 million peptoids. We believe that this is the first evidence that it is possible to develop sero-diagnostic assays - for any infectious disease - based on screening random libraries of non-biological molecular shapes. PMID:27182050

  8. Library+

    ERIC Educational Resources Information Center

    Merrill, Alex

    2011-01-01

    This article discusses possible future directions for academic libraries in the post Web/Library 2.0 world. These possible directions include areas such as data literacy, linked data sets, and opportunities for libraries in support of digital humanities. The author provides a brief sketch of the background information regarding the topics and…

  9. 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.

  10. Manipulation of unfolding-induced protein aggregation by peptides selected for aggregate-binding ability through phage display library screening.

    PubMed

    Kundu, Bishwajit; Shukla, Anshuman; Guptasarma, Purnananda

    2002-03-01

    A phage-displayed library of peptides (12-mer) was screened for the ability to bind to thermally aggregated bovine carbonic anhydrase (BCA), with a view toward examining whether peptides possessing this ability might bind to partially structured intermediates on the protein's unfolding pathway and, therefore, constitute useful tools for manipulation of the kinetic partitioning of molecules between the unfolded and aggregated states. Two peptides [N-HPSTMGLRTMHP-C and N-TPSAWKTALVKA-C] were identified and tested. While neither showed thermal aggregation autonomously, both peptides individually elicited remarkable increases in the levels of thermal aggregation of BCA. A possible explanation is that both peptides bind to surfaces on molten BCA that are not directly involved in aggregation. Such binding could slow down interconversions between folded and unfolded states and stabilize aggregation-prone intermediate(s) to make them more prone to aggregation, while failing to achieve any steric prevention of aggregation. The approach has the potential of yielding useful aggregation-aiding/inhibiting agents, and may provide clues to whether amorphous aggregates are "immobilized" forms of folding intermediates. PMID:11866450

  11. Humanization of a phosphothreonine peptide-specific chicken antibody by combinatorial library optimization of the phosphoepitope-binding motif.

    PubMed

    Baek, Du-San; Kim, Yong-Sung

    2015-07-31

    Detection of protein phosphorylation at a specific residue has been achieved by using antibodies, which have usually been raised by animal immunization. However, there have been no reports of the humanization of phosphospecific non-human antibodies. Here, we report the humanization of a chicken pT231 antibody specific to a tau protein-derived peptide carrying the phosphorylated threonine at residue 231 (pT231 peptide) as a model for better understanding the phosphoepitope recognition mechanism. In the chicken antibody, the phosphate group of the pT231-peptide antigen is exclusively recognized by complementarity determining region 2 of the heavy chain variable domain (VH-CDR2). Simple grafting of six CDRs of the chicken antibody into a homologous human framework (FR) template resulted in the complete loss of pT231-peptide binding. Using a yeast surface-displayed combinatorial library with permutations of 11 FR residues potentially affecting CDR loop conformations, we identified 5 critical FR residues. The back mutation of these residues to the corresponding chicken residues completely recovered the pT231-peptide binding affinity and specificity of the humanized antibody. Importantly, the back mutation of the FR 76 residue of VH (H76) (Asn to Ser) was critical in preserving the pT231-binding motif conformation via allosteric regulation of ArgH71, which closely interacts with ThrH52 and SerH52a residues on VH-CDR2 to induce the unique phosphate-binding bowl-like conformation. Our humanization approach of CDR grafting plus permutations of FR residues by combinatorial library screening can be applied to other animal antibodies containing unique binding motifs on CDRs specific to posttranslationally modified epitopes. PMID:26036575

  12. School Libraries and Language Skills in Indian Primary Schools: A Randomized Evaluation of the Akshara Library Program. NBER Working Paper No. 18183

    ERIC Educational Resources Information Center

    Borkum, Evan; He, Fang; Linden, Leigh L.

    2012-01-01

    We conduct a randomized evaluation of a school library program on children's language skills. We find that the program had little impact on students' scores on a language test administered 16 months after implementation. The estimates are sufficiently precise to rule out effects larger than 0.13 and 0.11 standard deviations based on the 95 and 90…

  13. A Cell-Based Approach for the Biosynthesis/Screening of Cyclic Peptide Libraries against Bacterial Toxins

    SciTech Connect

    Camarero, J A; Kimura, R; Woo, Y; Cantor, J; Steenblock, E

    2007-10-24

    Available methods for developing and screening small drug-like molecules able to knockout toxins or pathogenic microorganisms have some limitations. In order to be useful, these new methods must provide high-throughput analysis and identify specific binders in a short period of time. To meet this need, we are developing an approach that uses living cells to generate libraries of small biomolecules, which are then screened inside the cell for activity. Our group is using this new, combined approach to find highly specific ligands capable of disabling anthrax Lethal Factor (LF) as proof of principle. Key to our approach is the development of a method for the biosynthesis of libraries of cyclic peptides, and an efficient screening process that can be carried out inside the cell.

  14. 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.

  15. Exploring the venom proteome of the western diamondback rattlesnake, Crotalus atrox, via snake venomics and combinatorial peptide ligand library approaches.

    PubMed

    Calvete, Juan J; Fasoli, Elisa; Sanz, Libia; Boschetti, Egisto; Righetti, Pier Giorgio

    2009-06-01

    We report the proteomic characterization of the venom of the medically important North American western diamondback rattlesnake, Crotalus atrox, using two complementary approaches: snake venomics (to gain an insight of the overall venom proteome), and two solid-phase combinatorial peptide ligand libraries (CPLL), followed by 2D electrophoresis and mass spectrometric characterization of in-gel digested protein bands (to capture and "amplify" low-abundance proteins). The venomics approach revealed approximately 24 distinct proteins belonging to 2 major protein families (snake venom metalloproteinases, SVMP, and serine proteinases), which represent 69.5% of the total venom proteins, 4 medium abundance families (medium-size disintegrin, PLA(2), cysteine-rich secretory protein, and l-amino acid oxidase) amounting to 25.8% of the venom proteins, and 3 minor protein families (vasoactive peptides, endogenous inhibitor of SVMP, and C-type lectin-like). This toxin profile potentially explains the cytotoxic, myotoxic, hemotoxic, and hemorrhagic effects evoked by C. atrox envenomation. Further, our results showing that C. atrox exhibits a similar level of venom variation as Sistrurus miliarius points to a "diversity gain" scenario in the lineage leading to the Sistrurus catenatus taxa. On the other hand, the two combinatorial hexapeptide libraries captured distinct sets of proteins. Although the CPLL-treated samples did not retain a representative venom proteome, protein spots barely, or not at all, detectable in the whole venom were enriched in the two CPLL-treated samples. The amplified low copy number C. atrox venom proteins comprised a C-type lectin-like protein, several PLA(2) molecules, PIII-SVMP isoforms, glutaminyl cyclase isoforms, and a 2-cys peroxiredoxin highly conserved across the animal kingdom. Peroxiredoxin and glutaminyl cyclase may participate, respectively, in redox processes leading to the structural/functional diversification of toxins, and in the N

  16. Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

    PubMed Central

    Toledo-Machado, Christina Monerat; Machado de Avila, Ricardo Andrez; NGuyen, Christophe; Granier, Claude; Bueno, Lilian Lacerda; Carneiro, Claudia Martins; Menezes-Souza, Daniel; Carneiro, Rubens Antonio; Chávez-Olórtegui, Carlos; Fujiwara, Ricardo Toshio

    2015-01-01

    ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL. PMID:25710003

  17. FTIR spectroscopy of alanine-based peptides: assignment of the amide I' modes for random coil and helix.

    PubMed

    Martinez, G; Millhauser, G

    1995-01-01

    Fourier transform infrared (FTIR) spectroscopy has been used to explore the thermal unfolding of three helical, alanine-based peptides. Each of the peptides follows the general sequence Ac-(AAAX)nA-NH2 where X is either Lys+ or Arg+ and n = 3 or 4. These particular peptides were chosen because they contain varying amounts of 3(10)- and alpha-helix. The amide I' bands for all three peptides, under helix forming conditions, are between 1632 and 1635 cm-1. These results are incongruous with the assignment for alpha-helices in proteins where amide I' bands are usually found above 1650 cm-1. At elevated temperatures, all the peptides exhibit amide I' bands of 1642 cm-1, which is the accepted value for random coil. Variable temperature spectra for the 4K peptide (n = 4, X = Lys+), which is the most alpha-helical of the three peptides at 1 degree C, reveal an isosbestic point suggesting a cooperative two-state unfolding transition. The other peptides, however, did not reveal an isosbestic point, thereby indicating the presence of an intermediate, perhaps 3(10)-helix, along the thermal unfolding pathway.

  18. Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase.

    PubMed Central

    Allawi, H T; Dong, F; Ip, H S; Neri, B P; Lyamichev, V I

    2001-01-01

    A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta-globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility. PMID:11233988

  19. Selection of SARS-Coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice

    SciTech Connect

    Yu Hua; Jiang Lifang . E-mail: jianglf909@yahoo.com.cn; Fang Danyun; Yan Huijun; Zhou Jingjiao; Zhou Junmei; Liang Yu; Gao Yang; Zhao, Wei; Long Beiguo

    2007-03-15

    Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.

  20. Types of pediatric diabetes mellitus defined by anti-islet autoimmunity and random C-peptide at diagnosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to test the hypothesis that anti-islet autoantibody expression and random serum C-peptide obtained at diagnosis define phenotypes of pediatric diabetes with distinct clinical features. We analyzed 607 children aged <19 yr consecutively diagnosed with diabetes after ex...

  1. Prediction of ligand-binding sites of proteins by molecular docking calculation for a random ligand library.

    PubMed

    Fukunishi, Yoshifumi; Nakamura, Haruki

    2011-01-01

    A new approach to predicting the ligand-binding sites of proteins was developed, using protein-ligand docking computation. In this method, many compounds in a random library are docked onto the whole protein surface. We assumed that the true ligand-binding site would exhibit stronger affinity to the compounds in the random library than the other sites, even if the random library did not include the ligand corresponding to the true binding site. We also assumed that the affinity of the true ligand-binding site would be correlated to the docking scores of the compounds in the random library, if the ligand-binding site was correctly predicted. We call this method the molecular-docking binding-site finding (MolSite) method. The MolSite method was applied to 89 known protein-ligand complex structures extracted from the Protein Data Bank, and it predicted the correct binding sites with about 80-99% accuracy, when only the single top-ranked site was adopted. In addition, the average docking score was weakly correlated to the experimental protein-ligand binding free energy, with a correlation coefficient of 0.44.

  2. Cleavage Specificity Analysis of Six Type II Transmembrane Serine Proteases (TTSPs) Using PICS with Proteome-Derived Peptide Libraries

    PubMed Central

    Béliveau, François; Leduc, Richard; Overall, Christopher M.

    2014-01-01

    Background Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors. Methodology/Principal Finding To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS). Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin) to simultaneously determine sequence preferences on the N-terminal non-prime (P) and C-terminal prime (P’) sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1′ position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived. Conclusions Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1′ positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity. PMID:25211023

  3. The use of phage display peptide libraries for basic and translational research.

    PubMed

    Brissette, Renee; Goldstein, Neil I

    2007-01-01

    Phage display is a molecular technique, whereby genes are displayed in a functional form on the outer surfaces of bacteriophages by fusion to viral coat proteins. The gene product is encoded by a plasmid contained within the virus, which can be recovered and sequenced, linking the genetic information to the function of the protein. Phage display offers a powerful tool for the identification of short peptides or single chain antibodies that can bind and regulate the function of target proteins. One major advantage of phage display lies in its ability to rapidly identify target-specific peptides with pharmacological activity as agonists or antagonists. PMID:18217687

  4. Going nuts for nuts? The trace proteome of a Cola drink, as detected via combinatorial peptide ligand libraries.

    PubMed

    D'Amato, Alfonsina; Fasoli, Elisa; Kravchuk, Alexander V; Righetti, Pier Giorgio

    2011-05-01

    The "invisible" proteome of a Cola drink, stated to be produced with a kola nut extract, has been investigated via capture with combinatorial peptide ligand libraries (CPLL). Indeed, a few proteins in the M(r) 15-20 kDa range could be identified by treating large beverage volumes (1 L) and performing the capture with CPLLs at very acidic pH values (pH 2.2) under conditions mimicking reverse-phase adsorption. Ascertaining the presence of proteins deriving from plant extracts has confirmed the genuineness of such beverage and suggests the possibility of certifying whether soft drinks present on the market are indeed made with vegetable extracts or only with artificial chemical flavoring.

  5. Going nuts for nuts? The trace proteome of a Cola drink, as detected via combinatorial peptide ligand libraries.

    PubMed

    D'Amato, Alfonsina; Fasoli, Elisa; Kravchuk, Alexander V; Righetti, Pier Giorgio

    2011-05-01

    The "invisible" proteome of a Cola drink, stated to be produced with a kola nut extract, has been investigated via capture with combinatorial peptide ligand libraries (CPLL). Indeed, a few proteins in the M(r) 15-20 kDa range could be identified by treating large beverage volumes (1 L) and performing the capture with CPLLs at very acidic pH values (pH 2.2) under conditions mimicking reverse-phase adsorption. Ascertaining the presence of proteins deriving from plant extracts has confirmed the genuineness of such beverage and suggests the possibility of certifying whether soft drinks present on the market are indeed made with vegetable extracts or only with artificial chemical flavoring. PMID:21452894

  6. Definition of peptide inhibitors from a synthetic peptide library by targeting gelatinase B/matrix metalloproteinase-9 (MMP-9) and TNF-α converting enzyme (TACE/ADAM-17).

    PubMed

    Qiu, Zheng; Yan, Ming; Li, Qian; Liu, Datao; Van den Steen, Philippe E; Wang, Min; Opdenakker, Ghislain; Hu, Jialiang

    2012-08-01

    Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a regulatory and effector metalloproteinase in inflammation. TNF-α is an important proinflammatory cytokine and is released by the action of a Zn(2+)-containing converting enzyme (TACE/ADAM-17). Both metallo-enzymes play important roles during the development of shock syndromes. Combinatorial chemical synthesis and subsequent library deconvolution were previously used to define a peptide inhibitor (Regasepin1) acting, almost to the same degree, on neutrophil collagenase/MMP-8 and MMP-9 in vitro, and protecting mice against lethal endotoxinemia in vivo. We have now extended this approach by incorporating D-form amino acids and residues preferred by TACE. A new peptide library was designed and synthesized, and by deconvolution new peptide inhibitors were defined. These included a TACE-specific inhibitor, an MMP-9- specific inhibitor, and inhibitors for both enzymes.

  7. Molecular dynamics simulations of spontaneous fibril formation by random-coil peptides

    NASA Astrophysics Data System (ADS)

    Nguyen, Hung D.; Hall, Carol K.

    2004-11-01

    Assembly of normally soluble proteins into amyloid fibrils is a cause or associated symptom of numerous human disorders, including Alzheimer's and the prion diseases. We report molecular-level simulation of spontaneous fibril formation. Systems containing 12-96 model polyalanine peptides form fibrils at temperatures greater than a critical temperature that decreases with peptide concentration and exceeds the peptide's folding temperature, consistent with experimental findings. Formation of small amorphous aggregates precedes ordered nucleus formation and subsequent rapid fibril growth through addition of -sheets laterally and monomeric peptides at fibril ends. The fibril's structure is similar to that observed experimentally. amyloid | protein aggregation

  8. Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project.

    PubMed

    Park, Tae-Ho; Park, Beom-Seok; Kim, Jin-A; Hong, Joon Ki; Jin, Mina; Seol, Young-Joo; Mun, Jeong-Hwan

    2011-01-01

    As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome (BAC) by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones. Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa.

  9. Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project.

    PubMed

    Park, Tae-Ho; Park, Beom-Seok; Kim, Jin-A; Hong, Joon Ki; Jin, Mina; Seol, Young-Joo; Mun, Jeong-Hwan

    2011-01-01

    As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome (BAC) by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones. Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa. PMID:21338952

  10. Analysis and Prediction of the Critical Regions of Antimicrobial Peptides Based on Conditional Random Fields

    PubMed Central

    Chang, Kuan Y.; Lin, Tung-pei; Shih, Ling-Yi; Wang, Chien-Kuo

    2015-01-01

    Antimicrobial peptides (AMPs) are potent drug candidates against microbes such as bacteria, fungi, parasites, and viruses. The size of AMPs ranges from less than ten to hundreds of amino acids. Often only a few amino acids or the critical regions of antimicrobial proteins matter the functionality. Accurately predicting the AMP critical regions could benefit the experimental designs. However, no extensive analyses have been done specifically on the AMP critical regions and computational modeling on them is either non-existent or settled to other problems. With a focus on the AMP critical regions, we thus develop a computational model AMPcore by introducing a state-of-the-art machine learning method, conditional random fields. We generate a comprehensive dataset of 798 AMPs cores and a low similarity dataset of 510 representative AMP cores. AMPcore could reach a maximal accuracy of 90% and 0.79 Matthew’s correlation coefficient on the comprehensive dataset and a maximal accuracy of 83% and 0.66 MCC on the low similarity dataset. Our analyses of AMP cores follow what we know about AMPs: High in glycine and lysine, but low in aspartic acid, glutamic acid, and methionine; the abundance of α-helical structures; the dominance of positive net charges; the peculiarity of amphipathicity. Two amphipathic sequence motifs within the AMP cores, an amphipathic α-helix and an amphipathic π-helix, are revealed. In addition, a short sequence motif at the N-terminal boundary of AMP cores is reported for the first time: arginine at the P(-1) coupling with glycine at the P1 of AMP cores occurs the most, which might link to microbial cell adhesion. PMID:25803302

  11. Screening One-Bead-One-Compound Peptide Libraries for Optimal Kinase Substrates.

    PubMed

    Trinh, Thi B; Pei, Dehua

    2016-01-01

    Protein kinases phosphorylate specific serine, threonine, and/or tyrosine residues in their target proteins, resulting in functional changes of the target proteins such as enzymatic activity, cellular location, or association with other proteins. For many kinases, their in vivo substrate specificity is at least partially defined by the amino acid sequence surrounding the phosphorylatable residue (or sequence specificity). We report here a robust, high-throughput method for profiling the sequence specificity of protein kinases. Up to 10(7) different peptides are rapidly synthesized on PEGA beads in the one-bead-one-compound format and subjected to kinase reaction in the presence of [γ-S]ATP. Positive beads displaying the optimal kinase substrates are identified by covalently labeling the thiophosphorylated peptides with a fluorescent dye via a disulfide exchange reaction. Finally, the most active hit(s) is identified by the partial Edman degradation-mass spectrometry (PED-MS) method. The ability of this method to provide individual sequences of the preferred substrates permits the identification of sequence contextual effects and non-permissive residues. This method is applicable to protein serine, threonine, and tyrosine kinases. PMID:26501910

  12. Analysis of the immune response against mixotope peptide libraries from a main antigenic site of foot-and-mouth disease virus.

    PubMed

    Oliveira, Eliandre de; Jiménez-Clavero, Miguel Angel; Núñez, José Ignacio; Sobrino, Francisco; Andreu, David

    2005-04-01

    The design of vaccines for RNA viral diseases is complicated by the high genetic variability of the viruses, which favors the selection of escape mutants. A case in point is foot-and-mouth disease virus (FMDV), for which only limited protection has been observed in vaccination with single peptides. We have explored the potential of immunogens of higher sequence diversity, covering a broad range of field or culture-induced mutations at the immunodominant site A of FMDV, serotype C. Four mixotope-type peptide libraries, containing ca. 3 x 10(3) or ca. 3 x 10(5) peptides each, in either linear or cyclic form, and combining most significant mutations found or induced at site A have been synthesized and used to immunize guinea-pigs. Substantial levels of serum conversion have been observed for all four mixotope libraries, as well as for single peptides, linear or cyclic, corresponding to the consensus site A sequence. The specificity and neutralizing ability of the anti-mixotope and -peptide antibodies have been evaluated by direct ELISA and by plaque reduction and micro-neutralization assays, respectively. Challenge experiments with an infectious, guinea-pig-adapted FMDV strain, have shown higher protection rates in animals immunized with the cyclic versions, either in single sequence or in combinatorial mixotope form. PMID:15780448

  13. Identification of olive (Olea europaea) seed and pulp proteins by nLC-MS/MS via combinatorial peptide ligand libraries.

    PubMed

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

    2012-04-18

    Different types of extraction protocols are described for identifying proteins in seed and pulp of olive (Olea europea), by employing both conventional extraction methods and capture with ProteoMiner as well as with in house-made combinatorial peptide ligand libraries (HM-CPLLs) at pH 7.4 and at pH 2.2. Thanks to the use of CPLLs, able to dramatically amplify the signal of low-abundance species, a quite large number of compounds has been indeed identified: 61 in the seed (vs. only four reported in current literature) and 231 in the pulp (vs. 56 described so far), the deepest investigation up to the present of the olive proteome. In the seed, it highlights the presence of seed storage proteins, oleosins and histones. In the pulp, the allergenic thaumatin-like protein (Ole e 13) was confirmed, among the other 231, as the most abundant protein in the olive pulp. The present research has also been undertaken with the aim of identifying proteins in olive oil and ascertaining the relative contribution of seed and pulp proteins in their presence, if any, in oils.

  14. In-depth proteomic analysis of non-alcoholic beverages with peptide ligand libraries. I: Almond milk and orgeat syrup.

    PubMed

    Fasoli, Elisa; D'Amato, Alfonsina; Kravchuk, Alexander V; Citterio, Attilio; Righetti, Pier Giorgio

    2011-06-10

    Combinatorial peptide ligand libraries, both commercial and home-made, have been adopted to investigate the proteome of non-alcoholic beverages, in order to assess their genuineness and detect also trace proteins, in search of potential allergens. Two such beverages have been studied: almond milk and orgeat syrup. In the first product we have been able to identify 132 unique protein species, the deepest investigation so far of the almond proteome. In the second beverage, a handful of proteins (just 14) have been detected, belonging to a bitter almond extract. In both cases, the genuineness of such products has been verified, as well as the fact that almond milk, judging on the total protein and fat content, must have been produced with 100g ground almonds per litre of beverage, as required by authorities. On the contrary, cheap orgeat syrups produced by local supermarkets and sold as their own brands, where found not to contain any residual proteins, suggesting that they contained only synthetic aromas and no natural plant extracts. This could be the starting point for investigating the myriad of beverages that in the last decades have invaded the shelves of supermarkets the world over, whose genuineness and natural origin have never been properly assessed.

  15. Proteomic Analysis of Lonicera japonica Thunb. Immature Flower Buds Using Combinatorial Peptide Ligand Libraries and Polyethylene Glycol Fractionation.

    PubMed

    Zhu, Wei; Xu, Xiaobao; Tian, Jingkui; Zhang, Lin; Komatsu, Setsuko

    2016-01-01

    Lonicera japonica Thunb. flower is a well-known medicinal plant that has been widely used for the treatment of human disease. To explore the molecular mechanisms underlying the biological activities of L. japonica immature flower buds, a gel-free/label-free proteomic technique was used in combination with combinatorial peptide ligand libraries (CPLL) and polyethylene glycol (PEG) fractionation for the enrichment of low-abundance proteins and removal of high-abundance proteins, respectively. A total of 177, 614, and 529 proteins were identified in crude protein extraction, CPLL fractions, and PEG fractions, respectively. Among the identified proteins, 283 and 239 proteins were specifically identified by the CPLL and PEG methods, respectively. In particular, proteins related to the oxidative pentose phosphate pathway, signaling, hormone metabolism, and transport were highly enriched by CPLL and PEG fractionation compared to crude protein extraction. A total of 28 secondary metabolism-related proteins and 25 metabolites were identified in L. japonica immature flower buds. To determine the specificity of the identified proteins and metabolites for L. japonica immature flower buds, Cerasus flower buds were used, which resulted in the abundance of hydroxymethylbutenyl 4-diphosphate synthase in L. japonica immature flower buds being 10-fold higher than that in Cerasus flower buds. These results suggest that proteins related to secondary metabolism might be responsible for the biological activities of L. japonica immature flower buds.

  16. In-depth proteomic analysis of non-alcoholic beverages with peptide ligand libraries. I: Almond milk and orgeat syrup.

    PubMed

    Fasoli, Elisa; D'Amato, Alfonsina; Kravchuk, Alexander V; Citterio, Attilio; Righetti, Pier Giorgio

    2011-06-10

    Combinatorial peptide ligand libraries, both commercial and home-made, have been adopted to investigate the proteome of non-alcoholic beverages, in order to assess their genuineness and detect also trace proteins, in search of potential allergens. Two such beverages have been studied: almond milk and orgeat syrup. In the first product we have been able to identify 132 unique protein species, the deepest investigation so far of the almond proteome. In the second beverage, a handful of proteins (just 14) have been detected, belonging to a bitter almond extract. In both cases, the genuineness of such products has been verified, as well as the fact that almond milk, judging on the total protein and fat content, must have been produced with 100g ground almonds per litre of beverage, as required by authorities. On the contrary, cheap orgeat syrups produced by local supermarkets and sold as their own brands, where found not to contain any residual proteins, suggesting that they contained only synthetic aromas and no natural plant extracts. This could be the starting point for investigating the myriad of beverages that in the last decades have invaded the shelves of supermarkets the world over, whose genuineness and natural origin have never been properly assessed. PMID:21440098

  17. Proteomic Analysis of Lonicera japonica Thunb. Immature Flower Buds Using Combinatorial Peptide Ligand Libraries and Polyethylene Glycol Fractionation.

    PubMed

    Zhu, Wei; Xu, Xiaobao; Tian, Jingkui; Zhang, Lin; Komatsu, Setsuko

    2016-01-01

    Lonicera japonica Thunb. flower is a well-known medicinal plant that has been widely used for the treatment of human disease. To explore the molecular mechanisms underlying the biological activities of L. japonica immature flower buds, a gel-free/label-free proteomic technique was used in combination with combinatorial peptide ligand libraries (CPLL) and polyethylene glycol (PEG) fractionation for the enrichment of low-abundance proteins and removal of high-abundance proteins, respectively. A total of 177, 614, and 529 proteins were identified in crude protein extraction, CPLL fractions, and PEG fractions, respectively. Among the identified proteins, 283 and 239 proteins were specifically identified by the CPLL and PEG methods, respectively. In particular, proteins related to the oxidative pentose phosphate pathway, signaling, hormone metabolism, and transport were highly enriched by CPLL and PEG fractionation compared to crude protein extraction. A total of 28 secondary metabolism-related proteins and 25 metabolites were identified in L. japonica immature flower buds. To determine the specificity of the identified proteins and metabolites for L. japonica immature flower buds, Cerasus flower buds were used, which resulted in the abundance of hydroxymethylbutenyl 4-diphosphate synthase in L. japonica immature flower buds being 10-fold higher than that in Cerasus flower buds. These results suggest that proteins related to secondary metabolism might be responsible for the biological activities of L. japonica immature flower buds. PMID:26573373

  18. Combining rational and random strategies in β-glucosidase Zm-p60.1 protein library construction.

    PubMed

    Turek, Dušan; Klimeš, Pavel; Mazura, Pavel; Brzobohatý, Břetislav

    2014-01-01

    Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

  19. The comparison of BLyS-binding peptides from phage display library and computer-aided design on BLyS-TACI interaction.

    PubMed

    Zhao, Yacong; Hao, Xiafei; Feng, Jiannan; Shen, Beifen; Wei, Jing; Sun, Jian

    2015-02-01

    BLyS antagonists have become the therapeutic reagents in the treatment of autoimmune disorders. BLyS binding peptides and their Fc fusion proteins may be alternative BLyS antagonists in such application. In this study, the activity of BLyS binding peptide 814 obtained from phage display library and peptide TA designed by computer-aided modeling on the interaction of BLyS-TACI was compared. In addition, to maintain the spatial conformation and stability of the peptides, human IgG1 Fc fragment was fused to peptides 814 and TA to form peptide-Fc fusion proteins, steady and innovative peptibodies. The prokaryotic expression plasmids pET30a-814-Fc and pET30a-TA-Fc for these peptibodies were acquired by genetic engineering, and confirmed by DNA sequencing. After the right plasmids were transformed into Escherichia coli BL21 (DE3), the fusion proteins were expressed and purified by protein A affinity column. As a result of competitive ELISA, peptides 814 and TA at 100μg/ml displayed 52.2% and 28.6% inhibition on the interaction of TACI-Fc with BLyS respectively. Moreover, 814-Fc and TA-Fc fusion proteins could bind to BLyS in a dosage-dependent manner as TACI-Fc did, and displayed 54.7% and 26.1% inhibition on the interaction of TACI-Fc-Myc with BLyS at 100μg/ml respectively. So 814-Fc and TA-Fc proteins had the similar bioactivity as the peptides did. Furthermore, compared with TA-Fc, 814-Fc showed two-fold inhibition effect on BLyS binding to TACI, suggesting that 814-Fc could inhibit BLyS bioactivity significantly and might serve as a potential antagonist to treat autoimmune diseases associated with BLyS overexpression.

  20. Diversity and censoring of landscape phage libraries

    PubMed Central

    Kuzmicheva, G.A.; Jayanna, P.K.; Sorokulova, I.B.; Petrenko, V.A.

    2009-01-01

    Libraries of random peptides displayed on the surface of filamentous phages are a valuable source for biospecific ligands. However, their successful use can be hindered by a disproportionate representation of different phage clones and fluctuation of their composition that arises during phage reproduction, which have potential to affect efficiency of selection of clones with an optimal binding. Therefore, there is a need to develop phage display libraries with extended and varied repertoires of displayed peptides. In this work, we compared the complexity, evolution and representation of two phage display libraries displaying foreign octamers and nonamers in 4000 copies as the N-terminal part of the major coat protein pVIII of phage fd–tet (landscape libraries). They were obtained by replacement of amino acids 2–4 and 2–5 of pVIII with random octa- and nonamers, respectively. Statistical analysis of the libraries revealed their dramatic censoring and evolution during amplification. Further, a survey of both libraries for clones that bind common selectors revealed the presence of different non-overlapping families of target-specific clones in each library justifying the concept that different landscape libraries cover different areas of a sequence space. PMID:18988692

  1. Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

    SciTech Connect

    Dai, Xiaoyong; Cai, Cuizan; Xiao, Fei; Xiong, Yaoling; Huang, Yadong; Zhang, Qihao; Xiang, Qi; Lou, Guofeng; Lian, Mengyang; Su, Zhijian; Zheng, Qing

    2014-03-21

    Highlights: • A specific aFGF-binding peptide AP8 was identified from a phage display library. • AP8 could inhibit aFGF-stimulated cell proliferation in a dose-dependent manner. • AP8 arrested the cell cycle at the G0/G1 phase by suppressing Cyclin D1. • AP8 could block the activation of Erk1/2 and Akt kinase. • AP8 counteracted proliferation and cell cycle via influencing PA2G4 and PCNA. - Abstract: It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.

  2. 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 ...

  3. PLANET: a phage library analysis expert tool.

    PubMed

    Leplae, R; Tramontano, A

    1995-01-01

    In recent years random peptide libraries displayed on filamentous phage have been widely used and new ideas and techniques are continuously developing in the field (1-5). Notwithstanding this growing interest in the technique and in its promising results, and the enormous increase in usage and scope, very little effort has been devoted to the implementation of software able to handle and analyze the growing number of phage library-derived sequences. In our laboratory, phage libraries are extensively used and peptide sequences are continuously produced, so that the need arose of creating a database (6) to collect all the experimental results in a format compatible with GCG sequence analysis packages (7). We present here the description of an XWindow-based software package named PLANET (Phage Library ANalysis Expert Tool) devoted to the maintenance and statistical analysis of the database.

  4. Screening derivatized peptide libraries for tight binding inhibitors to carbonic anhydrase II by electrospray ionization-mass spectrometry.

    PubMed

    Gao, J; Cheng, X; Chen, R; Sigal, G B; Bruce, J E; Schwartz, B L; Hofstadler, S A; Anderson, G A; Smith, R D; Whitesides, G M

    1996-05-10

    This paper describes the use of electrospray ionization-mass spectrometry (ESI-MS) to screen two libraries of soluble compounds to search for tight binding inhibitors for carbonic anhydrase II (EC 4.2.1.1). The two libraries, H2NO2SC6H4C(O)NH-AA1-AA2-C(O)NHCH2CH2CO2H where AA1 and AA2 are L-amino acids (library size: 289 compounds) or D-amino acids (256 compounds), were constructed by attaching tripeptides to the carboxyl group of 4-carboxybenzenesulfonamide. Screening of both libraries yielded, as the tightest binding inhibitor, compound 1 (AA1 = AA2 = L-Leu; binding constant Kb = 1.4 x 10(8) M-1). The ability of ESI-MS to estimate simultaneously the relative binding affinities of a protein to soluble ligands in a library, if general, should be useful in drug development. PMID:8642553

  5. Topical Administration of a Connexin43-based peptide Augments Healing of Chronic Neuropathic Diabetic Foot Ulcers: A Multicenter, Randomized Trial

    PubMed Central

    Grek, Christina L.; Prasad, G.M.; Viswanathan, Vijay; Armstrong, David G.; Gourdie, Robert G.; Ghatnekar, Gautam S.

    2015-01-01

    Nonhealing neuropathic foot ulcers remain a significant problem in individuals with diabetes. The gap-junctional protein connexin43 (Cx43) has roles in dermal wound healing and targeting Cx43 signaling accelerates wound reepithelialization. In a prospective, randomized, multi-center clinical trial we evaluated the efficacy and safety of a peptide mimetic of the C-terminus of Cx43, ACT1, in accelerating the healing of chronic diabetic foot ulcers (DFUs) when incorporated into standard of care protocols. Adults with DFUs of at least four weeks duration were randomized to receive standard of care with or without topical application of ACT1. Primary outcome was mean percent ulcer reepithelialization and safety variables included incidence of treatment related adverse events and detection of ACT1 immunogenicity. ACT1 treatment was associated with a significantly greater reduction in mean percent ulcer area from baseline to 12 weeks (72.1% vs. 57.1%; p = 0.03). Analysis of incidence and median time-to-complete-ulcer closure revealed that ACT1 treatment was associated with a greater percentage of participants that reached 100% ulcer reepitheliazation and a reduced median time-to-complete-ulcer closure. No adverse events reported were treatment related, and ACT1 was not immunogenic. Treatment protocols that incorporate ACT1 may present a therapeutic strategy that safely augments the reepithelialization of chronic DFUs. PMID:25703647

  6. Mimotope peptides selected from phage display combinatorial library by serum antibodies of pigs experimentally infected with Taenia solium as leads to developing diagnostic antigens for human neurocysticercosis.

    PubMed

    Gazarian, Karlen; Rowlay, Merril; Gazarian, Tatiana; Vazquez Buchelli, Jorge Enrique; Hernández Gonzáles, Marisela

    2012-12-01

    Neurocysticercosis is caused by penetration of the tapeworm Taenia solium larvae into the central nervous system resulting in a diverse range of neurologic complications including epilepsy in endemic areas that globalization spreads worldwide. Sensitive and specific immunodiagnosis is needed for the early detection and elimination of the parasite, but the lack of standardized, readily obtainable antigens is a challenge. Here, we used the phage display for resolving the problem. The rationale of the strategy rests on the concept that the screening of combinatorial libraries with polyclonal serum to pathogens reveals families of peptides mimicking the pathogen most immunodominant epitopes indispensable for the successful diagnosis. The screening of a 7mer library with serum IgG of four pigs experimentally infected with parasite followed by computer aided segregation of the selected sequences resulted in the discovery of four clusters of homologous sequences of which one presented a family of ten mimotopes selected by three infected pig serum IgGs; the common motif sequence LSPF carried by the family was considered to be the core of an immunodominant epitope of the parasite critical for the binding with the antibody that selected the mimotopes. The immunoassay testing permitted to select a mimotope whose synthetic peptide free of the phage with the amino acid sequence Leu-Ser-Fen-Pro-Ser-Val-Val that distinguished well a panel of 21 cerebrospinal fluids of neurocysticercosis patients from the fluids of individuals with neurological complications of other etiology. This peptide is proposed as a lead for developing a novel molecularly defined diagnostic antigen(s) for the neurocysticercosis.

  7. Screening a phage display library for a novel FGF8b-binding peptide with anti-tumor effect on prostate cancer

    SciTech Connect

    Wang, Wenhui; Chen, Xilei; Li, Tao; Li, Yanmei; Wang, Ruixue; He, Dan; Luo, Wu; Li, Xiaokun; Wu, Xiaoping

    2013-05-01

    Fibroblast growth factor 8b (FGF8b) is the major isoform of FGF8 expressed in prostate cancer and it correlates with the stage and grade of the disease. FGF8b has been considered as a potential target for prostate cancer therapy. Here we isolated 12 specific FGF8b-binding phage clones by screening a phage display heptapeptide library with FGF8b. The peptide (HSQAAVP, named as P12) corresponding to one of these clones showed high homology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor (FGFR3c), contained 3 identical amino acids (AVP) to the authentic FGFR3 D2 sequence aa 163–169 (LLAVPAA) directly participating in ligand binding, carried the same charges as its corresponding motif (aa163–169) in FGFR3c, suggesting that P12 may have a greater potential to interrupt FGF8b binding to its receptors than other identified heptapeptides do. Functional analysis indicated that synthetic P12 peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrest cell cycle at the G0/G1 phase via suppression of Cyclin D1 and PCNA, and blockade of the activations of Erk1/2 and Akt cascades in both prostate cancer cells and vascular endothelial cells. The results demonstrated that the P12 peptide acting as an FGF8b antagonist may have therapeutic potential in prostate cancer. - Highlights: ► A novel FGF8b-binding peptide P12 was isolated from a phage display library. ► The mechanisms for P12 peptide inhibiting cell proliferation were proposed. ► P12 caused cell cycle arrest at G0/G1 phase via suppression of Cyclin D1 and PCNA. ► P12 suppressed FGF8b-induced activations of Akt and MAP kinases. ► P12 acting as an FGF8b antagonist may have therapeutic potential in prostate cancer.

  8. Density functional theory of equilibrium random copolymers: application to surface adsorption of aggregating peptides

    NASA Astrophysics Data System (ADS)

    Wang, Haiqiang; Forsman, Jan; Woodward, Clifford E.

    2016-06-01

    We generalize a recently developed polymer density functional theory (PDFT) for polydisperse polymer fluids to the case of equilibrium random copolymers. We show that the generalization of the PDFT to these systems allows us to obtain a remarkable simplification compared to the monodispersed polymers. The theory is used to treat a model for protein aggregation into linear filaments in the presence of surfaces. Here we show that, for attractive surfaces, there is evidence of significant enhancement of protein aggregation. This behaviour is a consequence of a surface phase transition, which has been shown to occur with ideal equilibrium polymers in the presence of sufficiently attractive surfaces. For excluding monomers, this transition is suppressed, though an echo of the underlying ideal transition is present in the sudden change in the excess adsorption.

  9. MEGAWHOP cloning: a method of creating random mutagenesis libraries via megaprimer PCR of whole plasmids.

    PubMed

    Miyazaki, Kentaro

    2011-01-01

    MEGAWHOP allows for the cloning of DNA fragments into a vector and is used for conventional restriction digestion/ligation-based procedures. In MEGAWHOP, the DNA fragment to be cloned is used as a set of complementary primers that replace a homologous region in a template vector through whole-plasmid PCR. After synthesis of a nicked circular plasmid, the mixture is treated with DpnI, a dam-methylated DNA-specific restriction enzyme, to digest the template plasmid. The DpnI-treated mixture is then introduced into competent Escherichia coli cells to yield plasmids carrying replaced insert fragments. Plasmids produced by the MEGAWHOP method are virtually free of contamination by species without any inserts or with multiple inserts, and also the parent. Because the fragment is usually long enough to not interfere with hybridization to the template, various types of fragments can be used with mutations at any site (either known or unknown, random, or specific). By using fragments having homologous sequences at the ends (e.g., adaptor sequence), MEGAWHOP can also be used to recombine nonhomologous sequences mediated by the adaptors, allowing rapid creation of novel constructs and chimeric genes. PMID:21601687

  10. Selection of a CXCR4 antagonist from a human heavy chain CDR3-derived phage library.

    PubMed

    Chevigné, Andy; Fischer, Aurélie; Mathu, Julie; Counson, Manuel; Beaupain, Nadia; Plesséria, Jean-Marc; Schmit, Jean-Claude; Deroo, Sabrina

    2011-08-01

    Phage display technology is a powerful selection approach to identify strong and specific binders to a large variety of targets. In this study, we compared the efficacy of a phage library displaying human heavy chain complementarity determining region 3 (HCDR3) repertoires with a set of conventional random peptide libraries for the identification of CXCR4 antagonists using a peptide corresponding to the second extracellular loop of the receptor CXCR4 as target. A total of 11 selection campaigns on this target did not result in any specific ligand from the random peptide libraries. In contrast, a single selection campaign with an HCDR3 library derived from the IgM repertoire of a nonimmunized donor resulted in nine specific peptides with lengths ranging from 10 to 19 residues. Four of these HCDR3 sequences interacted with native receptor and the most frequently isolated peptide displayed an affinity of 5.6 μm and acted as a CXCR4 antagonist (IC(50) = 23 μm). To comprehend the basis of the highly efficient HCDR3 library selection, its biochemical properties were investigated. The HCDR3 length varied from 3 to 21 residues and displayed a biased amino acid content with a predominant proportion of Tyr, Gly, Ser and Asp. Repetitive and conserved motifs were observed in the majority of the HCDR3 sequences. The strength and efficacy of the HCDR3 libraries reside in the combination of multiple size peptides and a naturally biased sequence variation. Therefore, HCDR3 libraries represent a powerful and versatile alternative to fully randomized peptide libraries, in particular for difficult targets.

  11. The Effect of a Connexin43-Based Peptide on the Healing of Chronic Venous Leg Ulcers: A Multicenter, Randomized Trial

    PubMed Central

    Ghatnekar, Gautam S; Grek, Christina L; Armstrong, David G; Desai, Sanjay C; Gourdie, Robert G

    2015-01-01

    The gap junction protein, connexin43 (Cx43), has critical roles in the inflammatory, edematous, and fibrotic processes following dermal injury and during wound healing, and is abnormally upregulated at the epidermal wound margins of venous leg ulcers (VLUs). Targeting Cx43 with ACT1, a peptide mimetic of the carboxyl-terminus of Cx43, accelerates fibroblast migration and proliferation, and wound reepithelialization. In a prospective, multicenter clinical trial conducted in India, adults with chronic VLUs were randomized to treatment with an ACT1 gel formulation plus conventional standard-of-care (SOC) protocols, involving maintaining wound moisture and four-layer compression bandage therapy, or SOC protocols alone. The primary end point was mean percent ulcer reepithelialization from baseline to 12 weeks. A significantly greater reduction in mean percent ulcer area from baseline to 12 weeks was associated with the incorporation of ACT1 therapy (79% (SD 50.4)) as compared with compression bandage therapy alone (36% (SD 179.8); P=0.02). Evaluation of secondary efficacy end points indicated a reduced median time to 50 and 100% ulcer reepithelialization for ACT1-treated ulcers. Incorporation of ACT1 in SOC protocols may represent a well-tolerated, highly effective therapeutic strategy that expedites chronic venous ulcer healing by treating the underlying ulcer pathophysiology through Cx43-mediated pathways. PMID:25072595

  12. Synthesis and screening of a CaaL peptide library versus FTase reveals a surprising number of substrates

    PubMed Central

    Krzysiak, Amanda J.; Aditya, Animesh V.; Hougland, James L.; Fierke, Carol A.; Gibbs, Richard A.

    2010-01-01

    Proteins bearing a CaaL sequence are typically geranylgeranylated to enable their proper localization and function. We found that many of the dansyl-GCaaL peptides representing mammalian CaaL proteins can be farnesylated by FTase. This result may have important implications for prenylated protein biology. PMID:20005705

  13. Monomeric Aβ1–40 and Aβ1–42 Peptides in Solution Adopt Very Similar Ramachandran Map Distributions That Closely Resemble Random Coil

    PubMed Central

    2016-01-01

    The pathogenesis of Alzheimer’s disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ1–40 and Aβ1–42 into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ1–42 compared to that of Aβ1–40 are poorly understood. To explore in detail the structural propensity of the monomeric Aβ1–40 and Aβ1–42 peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and J couplings. Systematic comparisons show that at neutral pH the Aβ1–40 and Aβ1–42 peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone J couplings (3JHNHα, 3JC′C′, 3JC′Hα, 1JHαCα, 2JNCα, and 1JNCα) recorded for Aβ1–40 were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ϕ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for αL at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D2O over H2O, particularly pronounced for Aβ1–42, suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process. PMID:26780756

  14. Monomeric Aβ(1-40) and Aβ(1-42) Peptides in Solution Adopt Very Similar Ramachandran Map Distributions That Closely Resemble Random Coil.

    PubMed

    Roche, Julien; Shen, Yang; Lee, Jung Ho; Ying, Jinfa; Bax, Ad

    2016-02-01

    The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ(1-40) and Aβ(1-42) into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ(1-42) compared to that of Aβ(1-40) are poorly understood. To explore in detail the structural propensity of the monomeric Aβ(1-40) and Aβ(1-42) peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and J couplings. Systematic comparisons show that at neutral pH the Aβ(1-40) and Aβ(1-42) peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone J couplings ((3)JHNHα, (3)JC'C', (3)JC'Hα, (1)JHαCα, (2)JNCα, and (1)JNCα) recorded for Aβ(1-40) were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ϕ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for αL at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D2O over H2O, particularly pronounced for Aβ(1-42), suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process.

  15. Glucagon-like peptide-1 receptor agonists versus insulin glargine for type 2 diabetes mellitus: A systematic review and meta-analysis of randomized controlled trials

    PubMed Central

    Li, Wei-Xin; Gou, Jian-Feng; Tian, Jin-Hui; Yan, Xiang; Yang, Lin

    2010-01-01

    Background: Glucagon-like peptide-1 (GLP-1) receptor agonists are a new class of hypoglycemic drugs, including exenatide, liraglutide, albiglutide, lixisenatide, and taspoglutide. Insulin glargine is a standard agent used to supplement basal insulin in type 2 diabetes mellitus (T2DM). Objective: The aim of this study was to review the efficacy and safety profiles of GLP-1 receptor agonists versus insulin glargine in type 2 diabetic patients who have not achieved treatment goals with oral hypoglycemic agents. Methods: The Cochrane Library, MEDLINE, EMBASE, Science Citation Index Expanded, and the database of ongoing trials were searched from inception through April 2010. Additional data were sought from relevant Web sites, the American Diabetes Association, reference lists of included trials and related (systematic) reviews, and industry. Randomized controlled trials (RCTs) were selected if they were ≥3 months in duration, compared GLP-1 receptor agonists with insulin glargine in patients with T2DM, and included ≥1 of the following outcomes: mortality, complications of T2DM, glycemie control, weight, lipids, blood pressure, adverse effects, and health-related quality of life. Quasirandomized controlled trials were excluded. The quality of the eligible studies was assessed on the basis of the following aspects: randomization procedure, allocation concealment, blinding, incomplete outcome data (intent-to-treat [ITT] analysis), selective outcome reporting, and publication bias. Results: A total of 410 citations were retrieved; 5 multicenter RCTs that met the inclusion criteria were identified. They were all open-label designs with an insulin glargine arm, predefined outcomes reported, and ITT analysis. One trial had an unclear randomization procedure and allocation concealment. Publication bias was not able to be determined. No data wete found with regard to mortality or diabetes-associated complications, and few data were found on quality of life. The results of

  16. A pH Sensitive High-Throughput Assay for miRNA Binding of a Peptide-Aminoglycoside (PA) Library.

    PubMed

    Watkins, Derrick; Jiang, Liuwei; Nahar, Smita; Maiti, Souvik; Arya, Dev P

    2015-01-01

    MicroRNAs (miRNA) are small RNAs that have a regulatory role in gene expression. Because of this regulatory role, miRNAs have become a new target for therapeutic compounds. Here, we outline an approach to target specific miRNAs using a high throughput capable assay and a 215 compound peptidic-aminosugar (PA) library. Aminosugars have been shown in a number of recent reports as important lead compounds that bind miRNA. In order to screen for compounds that bind miRNA, we have developed a high throughput displacement assay using a fluorescein-neomycin conjugated molecule (F-neo) as a probe for competitive miRNA binding compounds. We have applied the F-neo assay to four different miRNA constructs and the assay is applicable to most miRNAs, at various stages of processing. The results of the screen were validated by the determination of the IC50 for a select group of compounds from the library. For example, we identified eight compounds that bind to hsa-miR 504 with higher affinity than the parent neomycin. From the F-neo displacement assay we found that the number of binding sites differs for each miRNA, and the binding sites appear to differ both physically and chemically, with different affinity of the compounds resulting from the size of the molecule as well as the chemical structure. Additionally, the affinity of the compounds was dependent on the identity and position of the amino acid position of conjugation and the affinity of the compounds relative to other compounds in the library was miRNA dependent with the introduction of a second amino acid. PMID:26656788

  17. A pH Sensitive High-Throughput Assay for miRNA Binding of a Peptide-Aminoglycoside (PA) Library

    PubMed Central

    Watkins, Derrick; Jiang, Liuwei; Nahar, Smita; Maiti, Souvik; Arya, Dev P.

    2015-01-01

    MicroRNAs (miRNA) are small RNAs that have a regulatory role in gene expression. Because of this regulatory role, miRNAs have become a new target for therapeutic compounds. Here, we outline an approach to target specific miRNAs using a high throughput capable assay and a 215 compound peptidic-aminosugar (PA) library. Aminosugars have been shown in a number of recent reports as important lead compounds that bind miRNA. In order to screen for compounds that bind miRNA, we have developed a high throughput displacement assay using a fluorescein-neomycin conjugated molecule (F-neo) as a probe for competitive miRNA binding compounds. We have applied the F-neo assay to four different miRNA constructs and the assay is applicable to most miRNAs, at various stages of processing. The results of the screen were validated by the determination of the IC50 for a select group of compounds from the library. For example, we identified eight compounds that bind to hsa-miR 504 with higher affinity than the parent neomycin. From the F-neo displacement assay we found that the number of binding sites differs for each miRNA, and the binding sites appear to differ both physically and chemically, with different affinity of the compounds resulting from the size of the molecule as well as the chemical structure. Additionally, the affinity of the compounds was dependent on the identity and position of the amino acid position of conjugation and the affinity of the compounds relative to other compounds in the library was miRNA dependent with the introduction of a second amino acid. PMID:26656788

  18. Probability-Based Pattern Recognition and Statistical Framework for Randomization: Modeling Tandem Mass Spectrum/Peptide Sequence False Match Frequencies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Estimating and controlling the frequency of false matches between a peptide tandem mass spectrum and candidate peptide sequences is an issue pervading proteomics research. To solve this problem, we designed an unsupervised pattern recognition algorithm for detecting patterns with various lengths fr...

  19. Construction of a cDNA library for sea cucumber Acaudina leucoprocta and differential expression of ferritin peptide

    NASA Astrophysics Data System (ADS)

    Zhou, Jun; Hou, Fujing; Li, Ye; Su, Xiurong; Li, Taiwu; Jin, Chunhua

    2016-07-01

    Acaudina leucoprocta is an edible sea cucumber of economic interest that is widely distributed in China. Little information is available concerning the molecular genetics of this species although such knowledge would contribute to a better understanding of the optimal conditions for its aquaculture and its mechanisms of defense against disease. Therefore, we constructed a cDNA library and, based on bioinformatics analysis of the sequences, the functions of 75% of the cDNAs were identified, including those involved in cell structure, energy metabolism, mitochondrial function, and signal transduction pathways. Approximately 25% of genes in the library were unmatched. The gene for A. leucoprocta ferritin was also cloned. The predicted amino-acid sequence of ferritin displayed significant homology with other sea-cucumber counterparts but indicated that it was a new member of the ferritin family. Semiquantitative real-time RT-PCR indicated the highest levels of ferritin mRNA expression in the intestine. A polyclonal antibody of ferritin was also produced. These data provide a set of molecular tools essential for further studies of the functions of ferritin protein in A. leucoprocta.

  20. Mendelian Randomization Study of B-Type Natriuretic Peptide and Type 2 Diabetes: Evidence of Causal Association from Population Studies

    PubMed Central

    Pfister, Roman; Sharp, Stephen; Luben, Robert; Welsh, Paul; Barroso, Inês; Salomaa, Veikko; Meirhaeghe, Aline; Khaw, Kay-Tee; Sattar, Naveed; Langenberg, Claudia; Wareham, Nicholas J.

    2011-01-01

    Background Genetic and epidemiological evidence suggests an inverse association between B-type natriuretic peptide (BNP) levels in blood and risk of type 2 diabetes (T2D), but the prospective association of BNP with T2D is uncertain, and it is unclear whether the association is confounded. Methods and Findings We analysed the association between levels of the N-terminal fragment of pro-BNP (NT-pro-BNP) in blood and risk of incident T2D in a prospective case-cohort study and genotyped the variant rs198389 within the BNP locus in three T2D case-control studies. We combined our results with existing data in a meta-analysis of 11 case-control studies. Using a Mendelian randomization approach, we compared the observed association between rs198389 and T2D to that expected from the NT-pro-BNP level to T2D association and the NT-pro-BNP difference per C allele of rs198389. In participants of our case-cohort study who were free of T2D and cardiovascular disease at baseline, we observed a 21% (95% CI 3%–36%) decreased risk of incident T2D per one standard deviation (SD) higher log-transformed NT-pro-BNP levels in analysis adjusted for age, sex, body mass index, systolic blood pressure, smoking, family history of T2D, history of hypertension, and levels of triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. The association between rs198389 and T2D observed in case-control studies (odds ratio = 0.94 per C allele, 95% CI 0.91–0.97) was similar to that expected (0.96, 0.93–0.98) based on the pooled estimate for the log-NT-pro-BNP level to T2D association derived from a meta-analysis of our study and published data (hazard ratio = 0.82 per SD, 0.74–0.90) and the difference in NT-pro-BNP levels (0.22 SD, 0.15–0.29) per C allele of rs198389. No significant associations were observed between the rs198389 genotype and potential confounders. Conclusions Our results provide evidence for a potential causal role of the BNP

  1. 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

  2. Towards an Animal Model of Ovarian Cancer: Cataloging Chicken Blood Proteins Using Combinatorial Peptide Ligand Libraries Coupled with Shotgun Proteomic Analysis for Translational Research

    PubMed Central

    Ma, Yingying; Sun, Zeyu; de Matos, Ricardo; Zhang, Jing; Odunsi, Kunle

    2014-01-01

    Abstract Epithelial ovarian cancer is the most deadly gynecological cancer around the world, with high morbidity in industrialized countries. Early diagnosis is key in reducing its morbidity rate. Yet, robust biomarkers, diagnostics, and animal models are still limited for ovarian cancer. This calls for broader omics and systems science oriented diagnostics strategies. In this vein, the domestic chicken has been used as an ovarian cancer animal model, owing to its high rate of developing spontaneous epithelial ovarian tumors. Chicken blood has thus been considered a surrogate reservoir from which cancer biomarkers can be identified. However, the presence of highly abundant proteins in chicken blood has compromised the applicability of proteomics tools to study chicken blood owing to a lack of immunodepletion methods. Here, we demonstrate that a combinatorial peptide ligand library (CPLL) can efficiently remove highly abundant proteins from chicken blood samples, consequently doubling the number of identified proteins. Using an integrated CPLL-1DGE-LC-MSMS workflow, we identified a catalog of 264 unique proteins. Functional analyses further suggested that most proteins were coagulation and complement factors, blood transport and binding proteins, immune- and defense-related proteins, proteases, protease inhibitors, cellular enzymes, or cell structure and adhesion proteins. Semiquantitative spectral counting analysis identified 10 potential biomarkers from the present chicken ovarian cancer model. Additionally, many human homologs of chicken blood proteins we have identified have been independently suggested as diagnostic biomarkers for ovarian cancer, further triangulating our novel observations reported here. In conclusion, the CPLL-assisted proteomic workflow using the chicken ovarian cancer model provides a feasible platform for translational research to identify ovarian cancer biomarkers and understand ovarian cancer biology. To the best of our knowledge, we

  3. Towards an animal model of ovarian cancer: cataloging chicken blood proteins using combinatorial peptide ligand libraries coupled with shotgun proteomic analysis for translational research.

    PubMed

    Ma, Yingying; Sun, Zeyu; de Matos, Ricardo; Zhang, Jing; Odunsi, Kunle; Lin, Biaoyang

    2014-05-01

    Epithelial ovarian cancer is the most deadly gynecological cancer around the world, with high morbidity in industrialized countries. Early diagnosis is key in reducing its morbidity rate. Yet, robust biomarkers, diagnostics, and animal models are still limited for ovarian cancer. This calls for broader omics and systems science oriented diagnostics strategies. In this vein, the domestic chicken has been used as an ovarian cancer animal model, owing to its high rate of developing spontaneous epithelial ovarian tumors. Chicken blood has thus been considered a surrogate reservoir from which cancer biomarkers can be identified. However, the presence of highly abundant proteins in chicken blood has compromised the applicability of proteomics tools to study chicken blood owing to a lack of immunodepletion methods. Here, we demonstrate that a combinatorial peptide ligand library (CPLL) can efficiently remove highly abundant proteins from chicken blood samples, consequently doubling the number of identified proteins. Using an integrated CPLL-1DGE-LC-MSMS workflow, we identified a catalog of 264 unique proteins. Functional analyses further suggested that most proteins were coagulation and complement factors, blood transport and binding proteins, immune- and defense-related proteins, proteases, protease inhibitors, cellular enzymes, or cell structure and adhesion proteins. Semiquantitative spectral counting analysis identified 10 potential biomarkers from the present chicken ovarian cancer model. Additionally, many human homologs of chicken blood proteins we have identified have been independently suggested as diagnostic biomarkers for ovarian cancer, further triangulating our novel observations reported here. In conclusion, the CPLL-assisted proteomic workflow using the chicken ovarian cancer model provides a feasible platform for translational research to identify ovarian cancer biomarkers and understand ovarian cancer biology. To the best of our knowledge, we report here

  4. A randomized phase II trial of personalized peptide vaccine plus low dose estramustine phosphate (EMP) versus standard dose EMP in patients with castration resistant prostate cancer.

    PubMed

    Noguchi, Masanori; Kakuma, Tatsuyuki; Uemura, Hirotsugu; Nasu, Yasutomo; Kumon, Hiromi; Hirao, Yasuhiko; Moriya, Fukuko; Suekane, Shigetaka; Matsuoka, Kei; Komatsu, Nobukazu; Shichijo, Shigeki; Yamada, Akira; Itoh, Kyogo

    2010-07-01

    Personalized peptide vaccination (PPV) combined with chemotherapy could be a novel approach for many cancer patients. In this randomized study, we evaluated the anti-tumor effect and safety of PPV plus low-dose estramustine phosphate (EMP) as compared to standard-dose EMP for HLA-A2- or -A24-positive patients with castration resistant prostate cancer. Patients were randomized into groups receiving either PPV plus low-dose EMP (280 mg/day) or standard-dose EMP (560 mg/day). After disease progression, patients were switched to the opposite regime. The primary end point was progression-free survival (PFS). We randomly assigned 28 patients to receive PPV plus low-dose EMP and 29 patients to receive standard-dose EMP. Nineteen events in the PPV group and 20 events in the EMP group occurred during the first treatment. Median PFS for the first treatment was 8.5 months in the PPV group and 2.8 months in the EMP group with a hazard ratio (HR) of 0.28 (95% CI, 0.14-0.61; log-rank P = 0.0012), while there was no difference for median PFS for the second treatment. The HR for overall survival was 0.3 (95% CI, 0.1-0.91) in favor of the PPV plus low-dose EMP group (log-rank, P = 0.0328). The PPV plus low-dose EMP was well tolerated without major adverse effects and with increased levels of IgG and cytotoxic-T cell responses to the vaccinated peptides. PPV plus low-dose EMP was associated with an improvement in PSA-based PFS as compared to the standard-dose EMP alone.

  5. Facile Construction of Random Gene Mutagenesis Library for Directed Evolution Without the Use of Restriction Enzyme in Escherichia coli.

    PubMed

    Kim, Jae-Eung; Huang, Rui; Chen, Hui; You, Chun; Zhang, Y-H Percival

    2016-09-01

    A foolproof protocol was developed for the construction of mutant DNA library for directed protein evolution. First, a library of linear mutant gene was generated by error-prone PCR or molecular shuffling, and a linear vector backbone was prepared by high-fidelity PCR. Second, the amplified insert and vector fragments were assembled by overlap-extension PCR with a pair of 5'-phosphorylated primers. Third, full-length linear plasmids with phosphorylated 5'-ends were self-ligated with T4 ligase, yielding circular plasmids encoding mutant variants suitable for high-efficiency transformation. Self-made competent Escherichia coli BL21(DE3) showed a transformation efficiency of 2.4 × 10(5) cfu/µg of the self-ligated circular plasmid. Using this method, three mutants of mCherry fluorescent protein were found to alter their colors and fluorescent intensities under visible and UV lights, respectively. Also, one mutant of 6-phosphorogluconate dehydrogenase from a thermophilic bacterium Moorella thermoacetica was found to show the 3.5-fold improved catalytic efficiency (kcat /Km ) on NAD(+) as compared to the wild-type. This protocol is DNA-sequence independent, and does not require restriction enzymes, special E. coli host, or labor-intensive optimization. In addition, this protocol can be used for subcloning the relatively long DNA sequences into any position of plasmids. PMID:27367290

  6. Use of superparamagnetic beads for the isolation of a peptide with specificity to cymbidium mosaic virus.

    PubMed

    Ooi, Diana Jia Miin; Dzulkurnain, Adriya; Othman, Rofina Yasmin; Lim, Saw Hoon; Harikrishna, Jennifer Ann

    2006-09-01

    A modified method for the rapid isolation of specific ligands to whole virus particles is described. Biopanning against cymbidium mosaic virus was carried out with a commercial 12-mer random peptide display library. A solution phase panning method was devised using streptavidin-coated superparamagnetic beads. The solution based panning method was more efficient than conventional immobilized target panning when using whole viral particles of cymbidium mosaic virus as a target. Enzyme-linked immunosorbent assay of cymbidium mosaic virus-binding peptides isolated from the library identified seven peptides with affinity for cymbidium mosaic virus and one peptide which was specific to cymbidium mosaic virus and had no significant binding to odontoglossum ringspot virus. This method should have broad application for the screening of whole viral particles towards the rapid development of diagnostic reagents without the requirement for cloning and expression of single antigens.

  7. Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules

    PubMed Central

    Berkers, Celia R.; de Jong, Annemieke; Schuurman, Karianne G.; Linnemann, Carsten; Meiring, Hugo D.; Janssen, Lennert; Neefjes, Jacques J.; Schumacher, Ton N. M.; Rodenko, Boris

    2015-01-01

    Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I–restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags. PMID:26401003

  8. Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules.

    PubMed

    Berkers, Celia R; de Jong, Annemieke; Schuurman, Karianne G; Linnemann, Carsten; Meiring, Hugo D; Janssen, Lennert; Neefjes, Jacques J; Schumacher, Ton N M; Rodenko, Boris; Ovaa, Huib

    2015-11-01

    Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I-restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.

  9. Effect of pH, urea, peptide length, and neighboring amino acids on alanine alpha-proton random coil chemical shifts.

    PubMed

    Carlisle, Elizabeth A; Holder, Jessica L; Maranda, Abby M; de Alwis, Adamberage R; Selkie, Ellen L; McKay, Sonya L

    2007-01-01

    Accurate random coil alpha-proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, urea, peptide length and neighboring amino acids on the alpha-proton of Ala using model peptides of the general sequence GnXaaAYaaGn, where Xaa and Yaa are Leu, Val, Phe, Tyr, His, Trp or Pro, and n = 1-3. Changes in pH (2-6), urea (0-1M), and peptide length (n = 1-3) had no effect on Ala alpha-proton chemical shifts. Denaturing concentrations of urea (8M) caused significant downfield shifts (0.10 +/- 0.01 ppm) relative to an external DSS reference. Neighboring aliphatic residues (Leu, Val) had no effect, whereas aromatic amino acids (Phe, Tyr, His and Trp) and Pro caused significant shifts in the alanine alpha-proton, with the extent of the shifts dependent on the nature and position of the amino acid. Smaller aromatic residues (Phe, Tyr, His) caused larger shift effects when present in the C-terminal position (approximately 0.10 vs. 0.05 ppm N-terminal), and the larger aromatic tryptophan caused greater effects in the N-terminal position (0.15 ppm vs. 0.10 C-terminal). Proline affected both significant upfield (0.06 ppm, N-terminal) and downfield (0.25 ppm, C-terminal) chemical shifts. These new Ala correction factors detail the magnitude and range of variation in environmental chemical shift effects, in addition to providing insight into the molecular level interactions that govern protein folding.

  10. 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.

  11. Silent Encoding of Chemical Post-Translational Modifications in Phage-Displayed Libraries.

    PubMed

    Tjhung, Katrina F; Kitov, Pavel I; Ng, Simon; Kitova, Elena N; Deng, Lu; Klassen, John S; Derda, Ratmir

    2016-01-13

    In vitro selection of chemically modified peptide libraries presented on phage, while a powerful technology, is limited to one chemical post-translational modification (cPTM) per library. We use unique combinations of redundant codons to encode cPTMs with "silent barcodes" to trace multiple modifications within a mixed modified library. As a proof of concept, we produced phage-displayed peptide libraries Ser-[X]4-Gly-Gly-Gly, with Gly and Ser encoded using unique combinations of codons (TCA-[X]4-GGAGGAGGA, AGT-[X]4-GGTGGTGGT, etc., where [X]4 denotes a random NNK library). After separate chemical modification and pooling, mixed-modified libraries can be panned and deep-sequenced to identify the enriched peptide sequence and the accompanying cPTM simultaneously. We panned libraries bearing combinations of modifications (sulfonamide, biotin, mannose) against matched targets (carbonic anhydrase, streptavidin, concanavalin A) to identify desired ligands. Synthesis and validation of sequences identified by deep sequencing revealed that specific cPTMs are significantly enriched in panning against the specific targets. Panning on carbonic anhydrase yielded a potent ligand, sulfonamide-WIVP, with Kd = 6.7 ± 2.1 nM, a 20-fold improvement compared with the control ligand sulfonamide-GGGG. Silent encoding of multiple cPTMs can be readily incorporated into other in vitro display technologies such as bacteriophage T7 or mRNA display.

  12. Solution-phase parallel synthesis of a pharmacophore library of HUN-7293 analogues: a general chemical mutagenesis approach to defining structure-function properties of naturally occurring cyclic (depsi)peptides.

    PubMed

    Chen, Yan; Bilban, Melitta; Foster, Carolyn A; Boger, Dale L

    2002-05-15

    HUN-7293 (1), a naturally occurring cyclic heptadepsipeptide, is a potent inhibitor of cell adhesion molecule expression (VCAM-1, ICAM-1, E-selectin), the overexpression of which is characteristic of chronic inflammatory diseases. Representative of a general approach to defining structure-function relationships of such cyclic (depsi)peptides, the parallel synthesis and evaluation of a complete library of key HUN-7293 analogues are detailed enlisting solution-phase techniques and simple acid-base liquid-liquid extractions for isolation and purification of intermediates and final products. Significant to the design of the studies and unique to solution-phase techniques, the library was assembled superimposing a divergent synthetic strategy onto a convergent total synthesis. An alanine scan and N-methyl deletion of each residue of the cyclic heptadepsipeptide identified key sites responsible for or contributing to the biological properties. The simultaneous preparation of a complete set of individual residue analogues further simplifying the structure allowed an assessment of each structural feature of 1, providing a detailed account of the structure-function relationships in a single study. Within this pharmacophore library prepared by systematic chemical mutagenesis of the natural product structure, simplified analogues possessing comparable potency and, in some instances, improved selectivity were identified. One potent member of this library proved to be an additional natural product in its own right, which we have come to refer to as HUN-7293B (8), being isolated from the microbial strain F/94-499709.

  13. Acute ingestion of a novel whey-derived peptide improves vascular endothelial responses in healthy individuals: a randomized, placebo controlled trial

    PubMed Central

    Ballard, Kevin D; Bruno, Richard S; Seip, Richard L; Quann, Erin E; Volk, Brittanie M; Freidenreich, Daniel J; Kawiecki, Diana M; Kupchak, Brian R; Chung, Min-Yu; Kraemer, William J; Volek, Jeff S

    2009-01-01

    Background Whey protein is a potential source of bioactive peptides. Based on findings from in vitro experiments indicating a novel whey derived peptide (NOP-47) increased endothelial nitric oxide synthesis, we tested its effects on vascular function in humans. Methods A randomized, placebo-controlled, crossover study design was used. Healthy men (n = 10) and women (n = 10) (25 ± 5 y, BMI = 24.3 ± 2.3 kg/m2) participated in two vascular testing days each preceded by 2 wk of supplementation with a single dose of 5 g/day of a novel whey-derived peptide (NOP-47) or placebo. There was a 2 wk washout period between trials. After 2 wk of supplementation, vascular function in the forearm and circulating oxidative stress and inflammatory related biomarkers were measured serially for 2 h after ingestion of 5 g of NOP-47 or placebo. Macrovascular and microvascular function were assessed using brachial artery flow mediated dilation (FMD) and venous occlusion strain gauge plethysmography. Results Baseline peak FMD was not different for Placebo (7.7%) and NOP-47 (7.8%). Placebo had no effect on FMD at 30, 60, and 90 min post-ingestion (7.5%, 7.2%, and 7.6%, respectively) whereas NOP-47 significantly improved FMD responses at these respective postprandial time points compared to baseline (8.9%, 9.9%, and 9.0%; P < 0.0001 for time × trial interaction). Baseline reactive hyperemia forearm blood flow was not different for placebo (27.2 ± 7.2%/min) and NOP-47 (27.3 ± 7.6%/min). Hyperemia blood flow measured 120 min post-ingestion (27.2 ± 7.8%/min) was unaffected by placebo whereas NOP-47 significantly increased hyperemia compared to baseline (29.9 ± 7.8%/min; P = 0.008 for time × trial interaction). Plasma myeloperoxidase was increased transiently by both NOP-47 and placebo, but there were no changes in markers inflammation. Plasma total nitrites/nitrates significantly decreased over the 2 hr post-ingestion period and were lower at 120 min after placebo (-25%) compared to

  14. PNA-encoded chemical libraries.

    PubMed

    Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

    2015-06-01

    Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70.

  15. Uncovering the design rules for peptide synthesis of metal nanoparticles.

    PubMed

    Tan, Yen Nee; Lee, Jim Yang; Wang, Daniel I C

    2010-04-28

    Peptides are multifunctional reagents (reducing and capping agents) that can be used for the synthesis of biocompatible metal nanoparticles under relatively mild conditions. However, the progress in peptide synthesis of metal nanoparticles has been slow due to the lack of peptide design rules. It is difficult to establish sequence-reactivity relationships from peptides isolated from biological sources (e.g., biomineralizing organisms) or selected by combinatorial display libraries because of their widely varying compositions and structures. The abundance of random and inactive amino acid sequences in the peptides also increases the difficulty in knowledge extraction. In this study, a "bottom-up" approach was used to formulate a set of rudimentary rules for the size- and shape-controlled peptide synthesis of gold nanoparticles from the properties of the 20 natural alpha-amino acids for AuCl(4)(-) reduction and binding to Au(0). It was discovered that the reduction capability of a peptide depends on the presence of certain reducing amino acid residues, whose activity may be regulated by neighboring residues with different Au(0) binding strengths. Another finding is the effect of peptide net charge on the nucleation and growth of the Au nanoparticles. On the basis of these understandings, several multifunctional peptides were designed to synthesize gold nanoparticles in different morphologies (nanospheres and nanoplates) and with sizes tunable by the strategic placement of selected amino acid residues in the peptide sequence. The methodology presented here and the findings are useful for establishing the scientific basis for the rational design of peptides for the synthesis of metal nanostructures. PMID:20355728

  16. Screening and Antiviral Analysis of Phages That Display Peptides with an Affinity to Subunit C of Porcine Aminopeptidase

    PubMed Central

    Guo, Donghua; Zhu, Qinghe; Feng, Li

    2013-01-01

    The purified C subunit of the recombinant porcine aminopeptidase N (rpAPN-C) protein was used as an immobilized target to screen potential ligands against rpAPN-C from a 12-mer phage display random peptide library. After five rounds of biopanning, five phage clones showed specific binding affinities to rpAPN-C. In 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assays, the phage clone PM1, which contained the HDAISWTHYHPW peptide sequence, had a protective effect against TGEV infection in swine testis cells. Therefore, the HDAISWTHYHPW peptide sequence has a potential use as a small molecular therapeutic agent against TGEV infection. PMID:24111863

  17. Losing Libraries, Saving Libraries

    ERIC Educational Resources Information Center

    Miller, Rebecca

    2010-01-01

    This summer, as public libraries continued to get budget hit after budget hit across the country, several readers asked for a comprehensive picture of the ravages of the recession on library service. In partnership with 2010 Movers & Shakers Laura Solomon and Mandy Knapp, Ohio librarians who bought the Losing Libraries domain name, "LJ" launched…

  18. Biopanning of endotoxin-specific phage displayed peptides.

    PubMed

    Thomas, Celestine J; Sharma, Shilpi; Kumar, Gyanendra; Visweswariah, Sandhya S; Surolia, Avadhesha

    2003-07-18

    Systemic bacterial infections frequently lead to a plethora of symptoms termed "endotoxic shock" or "sepsis." Characterized by hypotension, coagulation abnormalities, and multiple organ failure, treatment of sepsis still remains mostly supportive. Of the various experimental therapeutic interventional strategies, neutralization of endotoxin by peptides or proteins is becoming popular recently. Hence, design of endotoxin binding peptides is gaining currency as their structural complexity and mode of recognition of endotoxin precludes mounting of resistance against them by the susceptible bacteria by genetic recombination, mutation, etc. Earlier work from our laboratory had shown that the amphiphilic cationic peptides are good ligands for endotoxin binding. In this study, we report the results of studies with the 12 selected lipid A binding phage displayed peptides by biopanning of a repertoire of a random pentadecapeptide library displayed on the filamentous M-13 phage. A comparison of the sequences revealed no consensus sequence between the 12 selected peptides suggesting that the lipid A binding motif is not sequence specific which is in accord with the sequence variation seen with the naturally occurring anti-microbial and/or endotoxin binding peptides. Thus, the flexibility of the peptides coupled with their plasticity in recognizing the lipid A moiety, explains their tight binding to endotoxin. At a structural level, asymmetric distribution of the charged polar residues on one face of the helix and non-polar residues on the opposite face appears to correlate with their activity.

  19. Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations

    PubMed Central

    Sun, Zhizeng; Mehta, Shrenik C.; Adamski, Carolyn J.; Gibbs, Richard A.; Palzkill, Timothy

    2016-01-01

    CphA is a Zn2+-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function. PMID:27616327

  20. Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.

    PubMed

    Sun, Zhizeng; Mehta, Shrenik C; Adamski, Carolyn J; Gibbs, Richard A; Palzkill, Timothy

    2016-01-01

    CphA is a Zn(2+)-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function. PMID:27616327

  1. Risk of bone fractures associated with glucagon-like peptide-1 receptor agonists' treatment: a meta-analysis of randomized controlled trials.

    PubMed

    Su, Bin; Sheng, Hui; Zhang, Manna; Bu, Le; Yang, Peng; Li, Liang; Li, Fei; Sheng, Chunjun; Han, Yuqi; Qu, Shen; Wang, Jiying

    2015-02-01

    Traditional anti-diabetic drugs may have negative or positive effects on risk of bone fractures. Yet the relationship between the new class glucagon-like peptide-1 receptor agonists (GLP-1 RA) and risk of bone fractures has not been established. We performed a meta-analysis including randomized controlled trials (RCT) to study the risk of bone fractures associated with liraglutide or exenatide, compared to placebo or other active drugs. We searched MEDLINE, EMBASE, and clinical trial registration websites for published or unpublished RCTs comparing the effects of liraglutide or exenatide with comparators. Only studies with disclosed bone fracture data were included. Separate pooled analysis was performed for liraglutide or exenatide, respectively, by calculating Mantel-Haenszel odds ratio (MH-OR). 16 RCTs were identified including a total of 11,206 patients. Liraglutide treatment was associated with a significant reduced risk of incident bone fractures (MH-OR=0.38, 95% CI 0.17-0.87); however, exenatide treatment was associated with an elevated risk of incident bone fractures (MH-OR=2.09, 95% CI 1.03-4.21). Publication bias and heterogeneity between studies were not observed. Our study demonstrated a divergent risk of bone fractures associated with different GLP-1 RA treatments. The current findings need to be confirmed by future well-designed prospective or RCT studies.

  2. The Synthetic Plasmodium falciparum Circumsporozoite Peptide PfCS102 as a Malaria Vaccine Candidate: A Randomized Controlled Phase I Trial

    PubMed Central

    Audran, Régine; Lurati-Ruiz, Floriana; Genton, Blaise; Blythman, Hildur E.; Ofori-Anyinam, Opokua; Reymond, Christophe; Corradin, Giampietro; Spertini, François

    2009-01-01

    Background Fully efficient vaccines against malaria pre-erythrocytic stage are still lacking. The objective of this dose/adjuvant-finding study was to evaluate the safety, reactogenicity and immunogenicity of a vaccine candidate based on a peptide spanning the C-terminal region of Plasmodium falciparum circumsporozoite protein (PfCS102) in malaria naive adults. Methodology and Principal Findings Thirty-six healthy malaria-naive adults were randomly distributed into three dose blocks (10, 30 and 100 µg) and vaccinated with PfCS102 in combination with either Montanide ISA 720 or GSK proprietary Adjuvant System AS02A at days 0, 60, and 180. Primary end-point (safety and reactogenicity) was based on the frequency of adverse events (AE) and of abnormal biological safety tests; secondary-end point (immunogenicity) on P. falciparum specific cell-mediated immunity and antibody response before and after immunization. The two adjuvant formulations were well tolerated and their safety profile was good. Most AEs were local and, when systemic, involved mainly fatigue and headache. Half the volunteers in AS02A groups experienced severe AEs (mainly erythema). After the third injection, 34 of 35 volunteers developed anti-PfCS102 and anti-sporozoite antibodies, and 28 of 35 demonstrated T-cell proliferative responses and IFN-γ production. Five of 22 HLA-A2 and HLA-A3 volunteers displayed PfCS102 specific IFN-γ secreting CD8+ T cell responses. Responses were only marginally boosted after the 3rd vaccination and remained stable for 6 months. For both adjuvants, the dose of 10 µg was less immunogenic in comparison to 30 and 100 µg that induced similar responses. AS02A formulations with 30 µg or 100 µg PfCS102 induced about 10-folds higher antibody and IFN-γ responses than Montanide formulations. Conclusions/Significance PfCS102 peptide was safe and highly immunogenic, allowing the design of more advanced trials to test its potential for protection. Two or three immunizations

  3. Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.

    SciTech Connect

    Rodi, D. J.; Soares, A. S.; Makowski, L.; Biosciences Division; BNL

    2002-01-01

    Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0{+-}1.6% of the random dodecapeptides and 7.9{+-}2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a {beta}-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.

  4. Targeting RNA with cysteine-constrained peptides

    PubMed Central

    Burns, Virginia A.; Bobay, Benjamin G.; Basso, Anne; Cavanagh, John; Melander, Christian

    2008-01-01

    A combined approach for targeting RNA with novel, biologically active ligands has been developed using a cyclic peptide library and in silico modeling. This approach has successfully identified novel cyclic peptide constructs that can target bTAR RNA. Subsequently, RNA/peptide interactions were effectively modeled using the HADDOCK docking program. PMID:18065222

  5. Artificial decoy spectral libraries for false discovery rate estimation in spectral library searching in proteomics.

    PubMed

    Lam, Henry; Deutsch, Eric W; Aebersold, Ruedi

    2010-01-01

    The challenge of estimating false discovery rates (FDR) in peptide identification from MS/MS spectra has received increased attention in proteomics. The simple approach of target-decoy searching has become popular with traditional sequence (database) searching methods, but has yet to be practiced in spectral (library) searching, an emerging alternative to sequence searching. We extended this target-decoy searching approach to spectral searching by developing and validating a robust method to generate realistic, but unnatural, decoy spectra. Our method involves randomly shuffling the peptide identification of each reference spectrum in the library, and repositioning each fragment ion peak along the m/z axis to match the fragment ions expected from the shuffled sequence. We show that this method produces decoy spectra that are sufficiently realistic, such that incorrect identifications are equally likely to match real and decoy spectra, a key assumption necessary for decoy counting. This approach has been implemented in the open-source library building software, SpectraST.

  6. A Combinatorial Peptide Ligand Libraries Treatment Followed by a Dual-Enzyme, Dual-Activation Approach on a nano-flow LC/Orbitrap/ETD for Comprehensive Analysis of Swine Plasma Proteome

    PubMed Central

    Tu, Chengjian; Li, Jun; Young, Rebeccah; Page, Brian J.; Engler, Frank; Halfon, Marc S.; Canty, John M.; Qu, Jun

    2011-01-01

    The plasma proteome holds enormous clinical potentials, yet an in-depth analysis of the plasma proteome remains a daunting challenge due to its high complexity and the extremely-wide dynamic range in protein concentrations. Furthermore, existing antibody-based approaches for depleting high-abundance proteins are not adaptable to the analysis of animal plasma proteome, which are often essential for experimental pathology/pharmacology. Here we describe a highly-comprehensive method for the investigation of animal plasma proteomes, which employs an optimized combinatorial peptide ligand libraries (CPLL) treatment to reduce the protein concentration dynamic range and a dual-enzyme, dual-activation strategy to achieve high proteomic coverage. The CPLL-treatment enriched the lower-abundance proteins by >100-fold when loading the samples in moderately-denaturing condition with multiple loading-washing cycles. The native and the CPLL-treated plasma were digested in-parallel respectively by two enzymes (trypsin and GluC) carrying orthogonal specificities. By performing this differential proteolysis, the proteome coverage is improved where peptides produced by only one enzyme are poorly detectable. Digests were fractionated with high-resolution SCX chromatography and then resolved on a long, heated nano-LC column. MS analysis was performed on an LTQ/Orbitrap respectively with two complementary activation methods (CID and ETD). We applied this optimized strategy to investigate the plasma proteome from swine, a prominent animal model for cardiovascular diseases(CVD). This large-scale analysis results in an identification of a total 3421 unique proteins, spanning a concentration range of 9–10 orders of magnitude. The proteins were identified under a set of commonly-accepted criteria including precursor mass error<15 ppm, Xcorr cutoffs, ≥two unique peptides at the peptide probability≥95% and protein probability≥99%, and the peptide FDR of the dataset was 1.8% as

  7. Two novel neutralizing antigenic epitopes of the s1 subunit protein of a QX-like avian infectious bronchitis virus strain Sczy3 as revealed using a phage display peptide library.

    PubMed

    Zou, Nianli; Xia, Jing; Wang, Fuyan; Duan, Zhenzhen; Miao, Dan; Yan, Qigui; Cao, Sanjie; Wen, Xintian; Liu, Ping; Huang, Yong

    2015-11-15

    The spike (S) protein of the infectious bronchitis virus (IBV) plays a central role in the pathogenicity, the immune antibody production, serotype and the tissue tropism. In this study, we generate 11 monoclonal antibodies (mAbs) against S1 subunit of IBV Sczy3 strain, and two mAbs 1D5 and 6A12 were positive in indirect ELISA against both His-S1 protein and the purified whole viral antigen. MAb 6A12 and 1D5 could recognized by other 10 IBV strains (IBVs) from five different genotypes, except that 1D5 had a relatively low reaction with two of the 10 tested IBVs. End-point neutralizing assay performed in chicken embro kidney (CEK) cells revealed that the neutralization titer of 6A12 and 1D5 against Sczy3 reached 1:44.7 and 1:40.6, respectively. After screening a phage display peptide library and peptide scanning, we identified two linear B-cell epitopes that were recognized by the mAbs 1D5 and 6A12, which corresponded to the amino acid sequences (87)PPQGMAW(93) and (412)IQTRTEP(418), respectively, in the IBV S1 subunit. Sequences comparison revealed that epitope (412)IQTRTEP(418) was conserved among IBVs, while the epitope (87)PPQGMAW(93) was relatively variable among IBVs. The novel mAbs and the epitopes identified will be useful for developing diagnostic assays for IBV infections.

  8. Creating Library Spaces: Libraries 2040.

    ERIC Educational Resources Information Center

    Bruijnzeels, Rob

    This paper suggests that by 2004, the traditional public libraries will have ceased to exist and new, attractive future libraries will have taken their place. The Libraries 2040 project of the Netherlands is initiating seven different libraries of the future. The Brabant library is the "ultimate library of the future" for the Dutch province of…

  9. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    NASA Astrophysics Data System (ADS)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  10. Biochemical functionalization of peptide nanotubes with phage displayed peptides.

    PubMed

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering. PMID:27479451

  11. Effects of Green Tea Extract on Insulin Resistance and Glucagon-Like Peptide 1 in Patients with Type 2 Diabetes and Lipid Abnormalities: A Randomized, Double-Blinded, and Placebo-Controlled Trial

    PubMed Central

    Liu, Chia-Yu; Huang, Chien-Jung; Huang, Lin-Huang; Chen, I-Ju; Chiu, Jung-Peng; Hsu, Chung-Hua

    2014-01-01

    The aim of this study is to investigate the effect of green tea extract on patients with type 2 diabetes mellitus and lipid abnormalities on glycemic and lipid profiles, and hormone peptides by a double-blinded, randomized and placebo-controlled clinical trial. This trial enrolled 92 subjects with type 2 diabetes mellitus and lipid abnormalities randomized into 2 arms, each arm comprising 46 participants. Of the participants, 39 in therapeutic arm took 500 mg green tea extract, three times a day, while 38 in control arm took cellulose with the same dose and frequency to complete the 16-week study. Anthropometrics measurements, glycemic and lipid profiles, safety parameters, and obesity-related hormone peptides were analyzed at screening and after 16-week course. Within-group comparisons showed that green tea extract caused a significant decrease in triglyceride and homeostasis model assessment of insulin resistance index after 16 weeks. Green tea extract also increased significantly high density lipoprotein cholesterol. The HOMA-IR index decreased from 5.4±3.9 to 3.5±2.0 in therapeutic arm only. Adiponectin, apolipoprotein A1, and apolipoprotein B100 increased significantly in both arms, but only glucagon-like peptide 1 increased in the therapeutic arm. However, only decreasing trend in triglyceride was found in between-group comparison. Our study suggested that green tea extract significantly improved insulin resistance and increased glucagon-like peptide 1 only in within-group comparison. The potential effects of green tea extract on insulin resistance and glucagon-like peptide 1 warrant further investigation. Trial Registration ClinicalTrials.gov NCT01360567 PMID:24614112

  12. Affinity-based release of polymer-binding peptides from hydrogels with the target segments of peptides.

    PubMed

    Serizawa, Takeshi; Fukuta, Hiroki; Date, Takaaki; Sawada, Toshiki

    2016-02-01

    Peptides with affinities for the target segments of polymer hydrogels were identified by biological screening using phage-displayed peptide libraries, and these peptides exhibited an affinity-based release capability from hydrogels. The results from cell culture assays demonstrated the sustained anticancer effects of the drug-conjugated peptides that were released from the hydrogels.

  13. Biodiscovery of aluminum binding peptides

    NASA Astrophysics Data System (ADS)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

    2013-05-01

    Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

  14. Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation

    SciTech Connect

    Nemoto, Naoto; Tsutsui, Chihiro; Yamaguchi, Junichi; Ueno, Shingo; Machida, Masayuki; Kobayashi, Toshikatsu; Sakai, Takafumi

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer Disulfide-rich peptide aptamer inhibits IL-6-dependent cell proliferation. Black-Right-Pointing-Pointer Disulfide bond of peptide aptamer is essential for its affinity to IL-6R. Black-Right-Pointing-Pointer Inhibitory effect of peptide depends on number and pattern of its disulfide bonds. -- Abstract: Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method 'cDNA display'. In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.

  15. Conformational Fine-Tuning of Pore-Forming Peptide Potency and Selectivity

    PubMed Central

    2015-01-01

    To better understand the sequence–structure–function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding–folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the “negative” peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity. PMID:26632653

  16. DIVERSE System: De Novo Creation of Peptide Tags for Non-enzymatic Covalent Labeling by In Vitro Evolution for Protein Imaging Inside Living Cells.

    PubMed

    Kawakami, Takashi; Ogawa, Koji; Goshima, Naoki; Natsume, Tohru

    2015-12-17

    Polypeptide-tag/small-molecule pairs for specific cellular protein labeling are useful for visualizing cellular proteins and controlling their activity. Here, we report the development of an in vitro evolution-based (poly)peptide tag identification system named the DIVERSE (Directed In Vitro Evolution of Reactive peptide tags via Sequential Enrichment) system. In this system, an extremely diverse (10(14)) library of peptide tags, displayed by covalent attachment to their encoding cDNAs, is continuously prepared from the DNA library in a one-pot approach. Using this system, we demonstrated de novo creation of non-enzymatically covalent-labeling peptide tags for a synthetic small-molecule target from a random peptide library. Protein labeling with these tags was applicable to N- and C-terminal fusions, multiple different proteins and fluorophores, and intracellular labeling. The DIVERSE system can be used not only for the de novo creation of polypeptide tags but also sequence optimization of existing polypeptide tags from extremely diverse libraries.

  17. Phage-display library biopanning and bioinformatic analysis yielded a high-affinity peptide to inflamed vascular endothelium both in vitro and in vivo.

    PubMed

    Yang, Min; Liu, Chenwu; Niu, Maochang; Hu, Yonghe; Guo, Mingyang; Zhang, Jun; Luo, Yong; Yuan, Weili; Yang, Mei; Yun, Mingdong; Guo, Linling; Yan, Jiao; Liu, Defang; Liu, Jinghua; Jiang, Yong

    2014-01-28

    Vascular inflammation is considered the primary pathological condition occurring in many chronic diseases. To detect the inflamed endothelium via imaging analysis or guide the drug to target lesions is therefore important for early diagnosis and treatment of vascular inflammatory diseases. In this study, we obtained a novel peptide NTTTH through high throughout biopanning and bioinformatic analysis. In vitro studies indicated that NTTTH homologs could especially target inflamed vascular endothelial cells, as imaging quantitative analysis indicated that the mean of integrated optical density (MIOD) and mean of stained area (MSA) were significantly higher versus control (P<0.05). In vivo studies showed that, after intravenous injection of enhanced green fluorescent protein (EGFP)-labeled NTTTH homologs into the lipopolysaccharide (LPS)-inflamed mice for 30min, NTTTH homologs were distributed in highly vascularized and inflamed organs like liver and kidney. As a control, little fluorescence could be detected in mice injected with EGFP alone. Cryosection showed that NTTTH homologs especially targeted inflamed vasculatures but not normal ones. We did not detect fluorescence signal in either normal or inflamed mice which were injected with EGFP alone. The results suggested the role of NTTTH homologs in guiding the targeted binding of EGFP to inflamed vasculature and the potential usage for imaging detection and drug delivery.

  18. Adoption of Library 2.0 Functionalities by Academic Libraries and Users: A Knowledge Management Perspective

    ERIC Educational Resources Information Center

    Kim, Yong-Mi; Abbas, June

    2010-01-01

    This study investigates the adoption of Library 2.0 functionalities by academic libraries and users through a knowledge management perspective. Based on randomly selected 230 academic library Web sites and 184 users, the authors found RSS and blogs are widely adopted by academic libraries while users widely utilized the bookmark function.…

  19. Peptide nanotubes.

    PubMed

    Hamley, Ian W

    2014-07-01

    The self-assembly of different classes of peptide, including cyclic peptides, amyloid peptides and surfactant-like peptides into nanotube structures is reviewed. The modes of self-assembly are discussed. Additionally, applications in bionanotechnology and synthetic materials science are summarized.

  20. Characterization of the substrate specificity of the major cysteine protease (cruzipain) from Trypanosoma cruzi using a portion-mixing combinatorial library and fluorogenic peptides.

    PubMed Central

    Nery, E D; Juliano, M A; Meldal, M; Svendsen, I; Scharfstein, J; Walmsley, A; Juliano, L

    1997-01-01

    The substrate specificity of the major cysteinyl proteinase of the parasitic protozoan Trypanosoma cruzi (cruzipain) was investigated, by combinatorial replacement of amino acid residues at positions P5-P'5, using a fluorescent quenched solid-phase library assay. Positively charged residues appear to be a general preference in the P5-P3 and the P'5-P'3 positions, while a hydrophobic residue was always required at the P2 position. A broad range of amino acids could be accepted at the P'1 position. A clear difference in terms of specificity between cruzipain and human cathepsin L was observed for the accommodation of Pro at the P2 position. The P1 specificity was investigated by a more detailed enzyme kinetic analysis using peptidyl-MCA (where MCA is methylcoumarin amide) and Abz-peptidyl-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] as substrates, and the results were compared with those obtained using human cathepsin L. Cruzipain showed a clear preference for benzyl-Cys or Arg at the P1 position. Human cathepsin L presented similar behaviour to that of cruzipain for the hydrolysis of the epsilon-NH2-Cap-Leu-Xaa-MCA (where Cap is epsilon-aminocaproyl) and Abz-Lys-Leu-Xaa-Phe-Ser-Lys-Gln-EDDnp series, whereas the mammalian enzyme was able to tolerate large P1 residues, such as phenylalanine, better than cruzipain in the latter series. PMID:9163334

  1. Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides

    PubMed Central

    Tal, Perry; Eizenberger, Shay; Cohen, Elad; Goldfinger, Naomi; Pietrokovski, Shmuel; Oren, Moshe; Rotter, Varda

    2016-01-01

    The p53 tumor suppressor serves as a major barrier against malignant transformation. Over 50% of tumors inactivate p53 by point mutations in its DNA binding domain. Most mutations destabilize p53 protein folding, causing its partial denaturation at physiological temperature. Thus a high proportion of human tumors overexpress a potential potent tumor suppressor in a non-functional, misfolded form. The equilibrium between the properly folded and misfolded states of p53 may be affected by molecules that interact with p53, stabilizing its native folding and restoring wild type p53 activity to cancer cells. To select for mutant p53 (mutp53) reactivating peptides, we adopted the phage display technology, allowing interactions between mutp53 and random peptide libraries presented on phages and enriching for phage that favor the correctly folded p53 conformation. We obtained a large database of potential reactivating peptides. Lead peptides were synthesized and analyzed for their ability to restore proper p53 folding and activity. Remarkably, many enriched peptides corresponded to known p53-binding proteins, including RAD9. Importantly, lead peptides elicited dramatic regression of aggressive tumors in mouse xenograft models. Such peptides might serve as novel agents for human cancer therapy. PMID:26943582

  2. A study of phenylalanine side-chain dynamics in surface-adsorbed peptides using solid-state deuterium NMR and rotamer library statistics.

    PubMed

    Li, Kun; Emani, Prashant S; Ash, Jason; Groves, Michael; Drobny, Gary P

    2014-08-13

    Extracellular matrix proteins adsorbed onto mineral surfaces exist in a unique environment where the structure and dynamics of the protein can be altered profoundly. To further elucidate how the mineral surface impacts molecular properties, we perform a comparative study of the dynamics of nonpolar side chains within the mineral-recognition domain of the biomineralization protein salivary statherin adsorbed onto its native hydroxyapatite (HAP) mineral surface versus the dynamics displayed by the native protein in the hydrated solid state. Specifically, the dynamics of phenylalanine side chains (viz., F7 and F14) located in the surface-adsorbed 15-amino acid HAP-recognition fragment (SN15: DpSpSEEKFLRRIGRFG) are studied using deuterium magic angle spinning ((2)H MAS) line shape and spin-lattice relaxation measurements. (2)H NMR MAS spectra and T1 relaxation times obtained from the deuterated phenylalanine side chains in free and HAP-adsorbed SN15 are fitted to models where the side chains are assumed to exchange between rotameric states and where the exchange rates and a priori rotameric state populations are varied iteratively. In condensed proteins, phenylalanine side-chain dynamics are dominated by 180° flips of the phenyl ring, i.e., the "π flip". However, for both F7 and F14, the number of exchanging side-chain rotameric states increases in the HAP-bound complex relative to the unbound solid sample, indicating that increased dynamic freedom accompanies introduction of the protein into the biofilm state. The observed rotameric exchange dynamics in the HAP-bound complex are on the order of 5-6 × 10(6) s(-1), as determined from the deuterium MAS line shapes. The dynamics in the HAP-bound complex are also shown to have some solution-like behavioral characteristics, with some interesting deviations from rotameric library statistics.

  3. Engineered Cystine-Knot Peptides That Bind αvβ3 Integrin With Antibody-Like Affinities

    PubMed Central

    Silverman, Adam P.; Levin, Aron M.; Lahti, Jennifer L.; Cochran, Jennifer R.

    2010-01-01

    The αvβ3 integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature, and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4 kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to αvβ3 integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a 6-amino acid loop of AgRP with a 9-amino acid loop containing the Arg-Gly-Asp (RGD) integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting (FACS) to identify clones with high affinity to detergent-solubilized αvβ3 integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing αvβ3 integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown bind specifically to αvβ3 integrins, and had only minimal or no binding to αvβ5, α5β1, and αiibβ3 integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally-occurring ligand for αvβ3 and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second generation libraries by individually randomizing these loops in one of the high affinity integrin-binding variants. Screening of these loop-randomized libraries against αvβ3 integrins resulted in peptides that retained high affinities for αvβ3 and had increased specificities for αvβ3 over αiibβ3 integrins. Collectively, these data

  4. Use of artificial intelligence in the design of small peptide antibiotics effective against a broad spectrum of highly antibiotic-resistant superbugs.

    PubMed

    Cherkasov, Artem; Hilpert, Kai; Jenssen, Håvard; Fjell, Christopher D; Waldbrook, Matt; Mullaly, Sarah C; Volkmer, Rudolf; Hancock, Robert E W

    2009-01-16

    Increased multiple antibiotic resistance in the face of declining antibiotic discovery is one of society's most pressing health issues. Antimicrobial peptides represent a promising new class of antibiotics. Here we ask whether it is possible to make small broad spectrum peptides employing minimal assumptions, by capitalizing on accumulating chemical biology information. Using peptide array technology, two large random 9-amino-acid peptide libraries were iteratively created using the amino acid composition of the most active peptides. The resultant data was used together with Artificial Neural Networks, a powerful machine learning technique, to create quantitative in silico models of antibiotic activity. On the basis of random testing, these models proved remarkably effective in predicting the activity of 100,000 virtual peptides. The best peptides, representing the top quartile of predicted activities, were effective against a broad array of multidrug-resistant "Superbugs" with activities that were equal to or better than four highly used conventional antibiotics, more effective than the most advanced clinical candidate antimicrobial peptide, and protective against Staphylococcus aureus infections in animal models.

  5. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders.

    PubMed

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A; de Greef, Tom F A; Abbaspourrad, Alireza; Weitz, David A; Chong, Shaorong

    2016-03-04

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (10(3)-10(6)). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

  6. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    PubMed Central

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

  7. National Libraries: An Analysis

    ERIC Educational Resources Information Center

    Burston, Godfrey

    1973-01-01

    The title National Library'' means different things in different countries. It can encompass parliamentary libraries, public libraries, special interest libraries (museums, etc.), and reference and lending libraries. (DH)

  8. Library Skills.

    ERIC Educational Resources Information Center

    Paul, Karin; Kuhlthau, Carol C.; Branch, Jennifer L.; Solowan, Diane Galloway; Case, Roland; Abilock, Debbie; Eisenberg, Michael B.; Koechlin, Carol; Zwaan, Sandi; Hughes, Sandra; Low, Ann; Litch, Margaret; Lowry, Cindy; Irvine, Linda; Stimson, Margaret; Schlarb, Irene; Wilson, Janet; Warriner, Emily; Parsons, Les; Luongo-Orlando, Katherine; Hamilton, Donald

    2003-01-01

    Includes 19 articles that address issues related to library skills and Canadian school libraries. Topics include information literacy; inquiry learning; critical thinking and electronic research; collaborative inquiry; information skills and the Big 6 approach to problem solving; student use of online databases; library skills; Internet accuracy;…

  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. Tumor-Penetrating Peptides

    PubMed Central

    Teesalu, Tambet; Sugahara, Kazuki N.; Ruoslahti, Erkki

    2013-01-01

    Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the integrin-binding RGD motif. RGD mediates tumor-homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular “zip code” of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies, and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is present in the

  11. Acyclic peptide inhibitors of amylases.

    PubMed

    Pohl, Nicola

    2005-12-01

    In this issue of Chemistry and Biology, a library screening approach reveals a linear octapeptide inhibitor of alpha-amylases reached by de novo design . The selected molecule shares characteristics with naturally occurring protein inhibitors -- a result that suggests general rules for the design of peptide-based amylase inhibitors may be achievable.

  12. Simple Cloning by Prolonged Overlap Extension-PCR with Application to the Preparation of Large-Size Random Gene Mutagenesis Library in Escherichia coli.

    PubMed

    Zhong, Chao; You, Chun; Wei, Ping; Zhang, Yi-Heng Percival

    2017-01-01

    We developed a simple method (simple cloning) for subcloning DNA fragments into any location of a targeted vector without the need of restriction enzyme, ligase, exonuclease, or recombinase in Escherichia coli. This technology can be applied to common E. coli hosts (e.g., DH5α, JM109, TOP10, BL21(DE3)). The protocol includes three steps: (1) generate DNA insert and linear vector backbone by regular high-fidelity PCR, where these two DNA fragments contain 3' and 5' overlapping termini; (2) generate DNA multimers based on these two DNA fragments by using prolonged overlap extension-PCR (POE-PCR) without primers added; and (3) transform POE-PCR product to competent Escherichia coli cells directly, yielding the desired plasmid. Simple cloning provides a new cloning method with great simplicity and flexibility. Furthermore, this new method can be modified for the preparation of a large-size mutant library for directed evolution in E. coli. Using this method, it is very easy to generate a mutant library with a size of more than 10(7) per 50 μL of the POE-PCR product within 1 day. PMID:27671931

  13. Simple Cloning by Prolonged Overlap Extension-PCR with Application to the Preparation of Large-Size Random Gene Mutagenesis Library in Escherichia coli.

    PubMed

    Zhong, Chao; You, Chun; Wei, Ping; Zhang, Yi-Heng Percival

    2017-01-01

    We developed a simple method (simple cloning) for subcloning DNA fragments into any location of a targeted vector without the need of restriction enzyme, ligase, exonuclease, or recombinase in Escherichia coli. This technology can be applied to common E. coli hosts (e.g., DH5α, JM109, TOP10, BL21(DE3)). The protocol includes three steps: (1) generate DNA insert and linear vector backbone by regular high-fidelity PCR, where these two DNA fragments contain 3' and 5' overlapping termini; (2) generate DNA multimers based on these two DNA fragments by using prolonged overlap extension-PCR (POE-PCR) without primers added; and (3) transform POE-PCR product to competent Escherichia coli cells directly, yielding the desired plasmid. Simple cloning provides a new cloning method with great simplicity and flexibility. Furthermore, this new method can be modified for the preparation of a large-size mutant library for directed evolution in E. coli. Using this method, it is very easy to generate a mutant library with a size of more than 10(7) per 50 μL of the POE-PCR product within 1 day.

  14. Estimating the diversity of peptide populations from limited sequence data.

    SciTech Connect

    Makowski, L.; Soares, A.; Biosciences Division; BNL

    2003-03-01

    Combinatorial libraries of peptides such as those displayed on the surface of a bacteriophage particle have become widely used tools for characterizing protein-protein and protein-small molecule interactions. The quality of a library frequently depends on its completeness, or diversity -- the proportion of possible sequences actually present in the library. The diversity of these libraries is frequently quoted on the basis of phage titers that provide little information about their completeness. Here, an analytical expression for diversity is introduced and a method for estimating the diversity of a peptide library from the sequences of a limited number of the members of the library is demonstrated. The diversities of a number of computationally constructed and actual peptide libraries are estimated using this method.

  15. Binding of Hemagglutinin and Influenza Virus to a Peptide-Conjugated Lipid Membrane

    PubMed Central

    Matsubara, Teruhiko; Shibata, Rabi; Sato, Toshinori

    2016-01-01

    Hemagglutinin (HA) plays an important role in the first step of influenza virus (IFV) infection because it initiates the binding of the virus to the sialylgalactose linkages of the receptors on the host cells. We herein demonstrate that a HA-binding peptide immobilized on a solid support is available to bind to HA and IFV. We previously obtained a HA-binding pentapeptide (Ala-Arg-Leu-Pro-Arg), which was identified by phage-display selection against HAs from random peptide libraries. This peptide binds to the receptor-binding site of HA by mimicking sialic acid. A peptide-conjugated lipid (pep-PE) was chemically synthesized from the peptide and a saturated phospholipid. A lipid bilayer composed of pep-PE and an unsaturated phospholipid (DOPC) was immobilized on a mica plate; and the interaction between HA and the pep-PE/DOPC membrane was investigated using atomic force microscopy. The binding of IFV to the pep-PE/DOPC membrane was detected by an enzyme-linked immunosorbent assay and real-time reverse transcription PCR. Our results indicate that peptide-conjugated lipids are a useful molecular device for the detection of HA and IFV. PMID:27092124

  16. Randomized Phase II Study of Docetaxel plus Personalized Peptide Vaccination versus Docetaxel plus Placebo for Patients with Previously Treated Advanced Wild Type EGFR Non-Small-Cell Lung Cancer.

    PubMed

    Takayama, Koichi; Sugawara, Shunichi; Saijo, Yasuo; Maemondo, Makoto; Sato, Atsushi; Takamori, Shinzo; Harada, Taishi; Sasada, Tetsuro; Kakuma, Tatsuyuki; Kishimoto, Junji; Yamada, Akira; Noguchi, Masanori; Itoh, Kyogo; Nakanishi, Yoichi

    2016-01-01

    Objectives. To evaluate the efficacy and safety of personalized peptide vaccination (PPV) combined with chemotherapy for patients with previously treated advanced non-small-cell lung cancer (NSCLC). Patients and Methods. Previously treated PS0-1 patients with IIIB/IV EGFR (epidermal growth factor receptor) wild genotype NSCLC were randomly assigned to docetaxel (60 mg/m(2) on Day 1) plus PPV based on preexisting host immunity or docetaxel plus placebo. Docetaxel administration was repeated every 3 weeks until disease progression. Personalized peptides or placebo was injected subcutaneously weekly in the first 8 weeks and biweekly in subsequent 16 weeks. The primary efficacy endpoint was progression-free survival (PFS). Results. PPV related toxicity was grade 2 or less skin reaction. The median PFS for placebo arm and PPV arm was 52 days and 59 days, respectively. There was no significant difference between two arms by log-rank test (p = 0.42). Interestingly, PFS and overall survival (OS) in humoral immunological responder were significantly longer than those in nonresponder. Conclusion. PPV did not improve the survival in combination with docetaxel for previously treated advanced NSCLC. However, PPV may be efficacious for the humoral immunological responders and a further clinical investigation is needed. PMID:27274999

  17. Peptide Aptamers That Bind to a Geminivirus Replication Protein Interfere with Viral Replication in Plant Cells †

    PubMed Central

    Lopez-Ochoa, Luisa; Ramirez-Prado, Jorge; Hanley-Bowdoin, Linda

    2006-01-01

    The AL1 protein of tomato golden mosaic virus (TGMV), a member of the geminivirus family, is essential for viral replication in plants. Its N terminus contains three conserved motifs that mediate origin recognition and DNA cleavage during the initiation of rolling-circle replication. We used the N-terminal domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide aptamer library constrained in the active site of the thioredoxin A (TrxA) gene. The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1 protein. Plant expression cassettes corresponding to the TrxA peptides and a TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and viral DNA replication was monitored by semiquantitative PCR. In these assays, 31 TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA levels to 13 to 64% of those of the wild type. All of the interfering aptamers also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the 20-mer peptides revealed that their sequences are not random. The alignments detected seven potential binding motifs, five of which are more highly represented among the interfering peptides. One motif was present in 18 peptides, suggesting that these peptides interact with a hot spot in the AL1 N terminus. The peptide aptamers characterized in these studies represent new tools for studying AL1 function and can serve as the basis for the development of crops with broad-based resistance to single-stranded DNA viruses. PMID:16731923

  18. Isolation of ZnO-binding 12-mer peptides and determination of their binding epitopes by NMR spectroscopy.

    PubMed

    Rothenstein, Dirk; Claasen, Birgit; Omiecienski, Beatrice; Lammel, Patricia; Bill, Joachim

    2012-08-01

    Inorganic-binding peptides are in the focus of research fields such as materials science, nanotechnology, and biotechnology. Applications concern surface functionalization by the specific coupling to inorganic target substrates, the binding of soluble molecules for sensing applications, or biomineralization approaches for the controlled formation of inorganic materials. The specific molecular recognition of inorganic surfaces by peptides is of major importance for such applications. Zinc oxide (ZnO) is an important semiconductor material which is applied in various devices. In this study the molecular fundamentals for a ZnO-binding epitope was determined. 12-mer peptides, which specifically bind to the zinc- or/and the oxygen-terminated sides of single-crystalline ZnO (0001) and (000-1) substrates, were selected from a random peptide library using the phage display technique. For two ZnO-binding peptides the mandatory amino acid residues, which are of crucial importance for the specific binding were determined with a label-free nuclear magnetic resonance (NMR) approach. NMR spectroscopy allows the identification of pH dependent interaction sites on the atomic level of 12-mer peptides and ZnO nanoparticles. Here, ionic and polar interaction forces were determined. For the oxygen-terminated side the consensus peptide-binding sequence (HSXXH) was predicted in silico and confirmed by the NMR approach. PMID:22720657

  19. Enzyme engineering by targeted libraries.

    PubMed

    Goldsmith, Moshe; Tawfik, Dan S

    2013-01-01

    This review outlines the strategies we apply for directed enzyme evolution using targeted libraries, namely, libraries that diversify specific residues with predefined mutational compositions. The theoretical grounds underlining the design of such libraries are described, including the mutational load, the ratio of beneficial versus deleterious mutations, and screening capacity. We point out the advantage of using mutational spiking strategies for "hedging the bets," exploring a large number of potentially beneficial mutations, and tuning the library's mutational load. Also highlighted are the merits of low-throughput screens that measure multiple parameters at high accuracy, and of using the desired substrate and reaction conditions rather than surrogates. We subsequently describe library construction strategies (rational and analytical) based on structure and sequence analyses, including ancestral libraries, which are particularly suitable for low-throughput screens. We also discuss the critical role of including compensatory, stabilizing mutations during library construction. Finally, the design efficiency and the optimal mutational loads of libraries are assessed by comparing targeted mutational libraries versus libraries of random mutations.

  20. Libraries program

    USGS Publications Warehouse

    2011-01-01

    The U.S. Congress authorized a library for the U.S. Geological Survey (USGS) in 1879. The library was formally established in 1882 with the naming of the first librarian and began with a staff of three and a collection of 1,400 books. Today, the USGS Libraries Program is one of the world's largest Earth and natural science repositories and a resource of national significance used by researchers and the public worldwide.

  1. America's Star Libraries: Top-Rated Libraries

    ERIC Educational Resources Information Center

    Lance, Keith Curry; Lyons, Ray

    2009-01-01

    "Library Journal"'s national rating of public libraries, the "LJ" Index of Public Library Service 2009, Round 2, identifies 258 "star" libraries. Created by Keith Curry Lance and Ray Lyons and based on 2007 data from the IMLS, it rates 7,268 public libraries. The top libraries in each group get five, four, or three stars. All included libraries,…

  2. Tandem mass spectrometry spectral libraries and library searching.

    PubMed

    Deutsch, Eric W

    2011-01-01

    Spectral library searching in the field of proteomics has been gaining visibility and use in the last few years, primarily due to the expansion of public proteomics data repositories and the large spectral libraries that can be generated from them. Spectral library searching has several advantages over conventional sequence searching: it is generally much faster, and has higher specificity and sensitivity. The speed increase is primarily, due to having a smaller, fully indexable search space of real spectra that are known to be observable. The increase in specificity and sensitivity is primarily due to the ability of a search engine to utilize the known intensities of the fragment ions, rather than just comparing with theoretical spectra as is done with sequence searching. The main disadvantage of spectral library searching is that one can only identify peptide ions that have been seen before and are stored in the spectral library. In this chapter, an overview of spectral library searching and the libraries currently available are presented.

  3. Privatizing Libraries

    ERIC Educational Resources Information Center

    Jerrard, Jane; Bolt, Nancy; Strege, Karen

    2012-01-01

    This timely special report from ALA Editions provides a succinct but comprehensive overview of the "privatization" of public libraries. It provides a history of the trend of local and state governments privatizing public services and assets, and then examines the history of public library privatization right up to the California legislation…

  4. Library Advocacy

    ERIC Educational Resources Information Center

    Plunkett, Kate

    2010-01-01

    This paper is about the issue of advocacy. Standing at the vanguard of literacy, library media specialists have a unique role. However, it is time for media specialists to advocate their services in a proactive way. If library media specialists cannot, both individually and collectively, put advocacy at the forefront, then students will suffer the…

  5. Library Legislation.

    ERIC Educational Resources Information Center

    Kavass, Igor

    Examination of several library legislation models developed to meet the needs of developed and developing nations reveals that our traditional notion of the library's role in society must be abandoned if we wish to reconcile its benefits to its costs. Four models currently exist: many nations, particularly Asian, have no legislation; most nations,…

  6. Chemical library screening using a SPR-based inhibition in solution assay: simulations and experimental validation.

    PubMed

    Choulier, Laurence; Nominé, Yves; Zeder-Lutz, Gabrielle; Charbonnier, Sebastian; Didier, Bruno; Jung, Marie-Louise; Altschuh, Danièle

    2013-09-17

    We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (KD = 0.8 μM) interaction between an E6-derived peptide (E6peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 μM) and limited peptide densities (Rmax < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 μM, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library® (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with KD in the 10-50 μM range. We propose that a medium (μM) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening. PMID:23931734

  7. Effects of the properties of short peptides conjugated with cell-penetrating peptides on their internalization into cells.

    PubMed

    Matsumoto, Ryo; Okochi, Mina; Shimizu, Kazunori; Kanie, Kei; Kato, Ryuji; Honda, Hiroyuki

    2015-01-01

    Peptides, especially intracellular functional peptides that can play a particular role inside a cell, have attracted attention as promising materials to control cell fate. However, hydrophilic materials like peptides are difficult for cells to internalize. Therefore, the screening and design of intracellular functional peptides are more difficult than that of extracellular ones. An effective high-throughput screening system for intracellular functional peptides has not been reported. Here, we demonstrate a novel peptide array system for screening intracellular functional peptides, in which both cell-penetrating peptide (CPP) domain and photo-cleavable linkers are used. By using this screening system, we determined how the cellular uptake properties of CPP-conjugated peptides varied depending on the properties of the conjugated peptides. We found that the internalization ability of CPP-conjugated peptides varied greatly depending on the property of the conjugated peptides, and anionic peptides drastically decreased the uptake ability. We summarized our data in a scatter diagram that plots hydrophobicity versus isoelectric point (pI) of conjugated peptides. These results define a peptide library suitable for screening of intracellular functional peptides. Thus, our system, including the diagram, is a promising tool for searching biological active molecules such as peptide-based drugs. PMID:26256261

  8. A novel bioactive peptide from wasp venom

    PubMed Central

    Chen, Lingling; Chen, Wenlin; Yang, Hailong; Lai, Ren

    2010-01-01

    Wasp venoms contain a number of pharmacologically active biomolecules, undertaking a wide range of functions necessary for the wasp's survival. We purified and characterized a novel bioactive peptide (vespin) from the venoms of Vespa magnifica (Smith) wasps with unique primary structure. Its amino acid sequence was determined to be CYQRRVAITAGGLKHRLMSSLIIIIIIRINYLRDNSVIILESSY. It has 44 residues including 15 leucines or isoleucines (32%) in the sequence. Vespin showed contractile activity on isolated ileum smooth muscle. The cDNA encoding vespin precursor was cloned from the cDNA library of the venomous glands. The precursor consists of 67 amino acid residues including the predicted signal peptide and mature vespin. A di-basic enzymatic processing site (-KR-) is located between the signal peptide and the mature peptide. Vespin did not show similarity with any known proteins or peptides by BLAST search, suggesting it is a novel bioactive peptide from wasp venoms. PMID:21544181

  9. [The Presidential Libraries.

    ERIC Educational Resources Information Center

    Webb, John

    There are seven Presidential libraries in various states of existence, from quite active to proposed: (1) Franklin D. Roosevelt Library, (2) Harry S. Truman Library, (3) Herbert Hoover Library, (4) Dwight D. Eisenhower Library, (5) John F. Kennedy Memorial Library (6) Lyndon B. Johnson Library and (7) Rutherford B. Hayes Memorial Library. Each…

  10. Development of a peptide by phage display for SPECT imaging of resistance-susceptible breast cancer

    PubMed Central

    Larimer, Benjamin M; Deutscher, Susan L

    2014-01-01

    Personalized medicine is at the forefront of cancer diagnosis and therapy. Molecularly targeted therapies such as trastuzumab and tamoxifen have enhanced prognosis of patients with cancers expressing ERBB2 and the estrogen receptor, respectively. One obstacle to targeted therapy is the development of resistance. A targeted peptide that could distinguish resistance-susceptible cancer would aid in treatment. BT-474 human breast cancer cells can be resistant to both tamoxifen and trastuzumab, and may serve as a model for malignancies in which targeted therapy may not work. Bacteriophage (phage) display is a combinatorial technology that has been used to isolate peptides that target a specific cancer subtype. It was hypothesized that in vivo phage display could be used to select a peptide for SPECT imaging of BT-474 human breast cancer xenografts. A phage library displaying random 15 amino acid peptides was subjected to four rounds of selection, after which 14 clones were analyzed for BT-474 binding and specificity. One phage clone, 51, demonstrated superior binding and specificity, and the displayed peptide was synthesized for in vitro characterization. Peptide 51 bound specifically to BT-474 cells with an EC50 = 2.33 µM and was synthesized as a DOTA-conjugated peptide and radiolabeled with 111In for in vitro and in vivo analysis. The radiolabeled peptide exhibited an IC50 = 16.1 nM to BT-474 cells and its biodistribution and SPECT imaging in BT-474 xenografted mice was analyzed. Although tumor uptake was moderate at 0.11% ID/g, SPECT imaging revealed a distinct tumor vasculature binding pattern. It was discovered that peptide 51 had an identical 5 amino acid N-terminal sequence to a peptide, V1, which bound to Nrp1, a tumor vasculature protein. Peptide 51 and V1 were examined for binding to target cells, and 51 bound both target and endothelial cells, while V1 only bound endothelial cells. Truncated versions of 51 did not bind BT-474 cells, demonstrating that the

  11. Standards for British Libraries.

    ERIC Educational Resources Information Center

    Vaughan, Anthony

    1982-01-01

    Reviews developments in British library standards since 1971, highlighting types of standards, public libraries, academic libraries (university, polytechnic, college), school libraries, and special libraries (hospital and health sciences, prison, subject specializations). Thirty-nine references are cited. (EJS)

  12. Digital Libraries.

    ERIC Educational Resources Information Center

    Fox, Edward A.; Urs, Shalini R.

    2002-01-01

    Provides an overview of digital libraries research, practice, and literature. Highlights include new technologies; redefining roles; historical background; trends; creating digital content, including conversion; metadata; organizing digital resources; services; access; information retrieval; searching; natural language processing; visualization;…

  13. Callpath Library

    2013-11-09

    The "Callpath Library" is a software abstraction layer over a number of stack tracing utilities. It allows tool develoopers to conveniently represent and mNipulate call paths gathered fro U. Wisconsin's Stackwalker API and GNU Backtrace.

  14. Peptide and non-peptide HIV fusion inhibitors.

    PubMed

    Jiang, Shibo; Zhao, Qian; Debnath, Asim K

    2002-01-01

    Fusion of the HIV envelope with the target cell membrane is a critical step of HIV entry into the target cell. The HIV envelope glycoprotein gp41 plays an important role in the fusion of viral and target cell membranes and serves as an attractive target for development of HIV fusion inhibitors. The extracellular domain of gp41 contains three important functional regions, i.e. fusion peptide (FP), N- and C-terminal heptad repeats (NHR and CHR, respectively). The FP region is composed of hydrophobic, glycine-rich residues that are essential for the initial penetration of the target cell membrane. NHR and CHR regions consist of hydrophobic residues, which have the tendency to form alpha-helical coiled coils. During the process of fusion of HIV or HIV-infected cells with uninfected cells, FP inserts into the target cell membrane and subsequently the NHR and CHR regions change conformations and associate with each other to form a fusion-active gp41 core. Peptides derived from NHR and CHR regions, designated N- and C-peptides, respectively, have potent inhibitory activity against HIV fusion by binding to the CHR and NHR regions, respectively, to prevent the formation of the fusion-active gp41 core. C-peptide may also bind to FP, thereby blocking its insertion into the target cell membrane. One of the C-peptides, T-20, which is in the phase III clinical trials, has potent in vivo activity against HIV infection and is expected to become the first peptide HIV fusion inhibitory drug in the near future. However, this peptide HIV fusion inhibitor lacks oral availability and is sensitive to the proteolytic digestion. Therefore, it is essential to develop small molecular non-peptide HIV fusion inhibitors having a mechanism of action similar to the C-peptides. One of the approaches in identifying the inhibitors is to use an immunological assay to screen chemical libraries for the compounds that potentially block the interaction between the NHR and CHR regions to form a fusion

  15. 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.

  16. Spectral library generating function for assessing spectrum-spectrum match significance.

    PubMed

    Wang, Mingxun; Bandeira, Nuno

    2013-09-01

    Tandem mass spectrometry (MS/MS) continues to be the technology of choice for high-throughput analysis of complex proteomics samples. While MS/MS spectra are commonly identified by matching against a database of known protein sequences, the complementary approach of spectral library searching against collections of reference spectra consistently outperforms sequence-based searches by resulting in significantly more identified spectra. However, while spectral library searches benefit from the advance knowledge of the expected peptide fragmentation patterns recorded in library spectra, estimation of the statistical significance of spectrum-spectrum matches (SSMs) continues to be hindered by difficulties in finding an appropriate definition of "random" SSMs to use as a null model when estimating the significance of true SSMs. We propose to avoid this problem by changing the null hypothesis: instead of determining the probability of observing a high SSM score between randomly matched spectra, we estimate the probability of observing a low SSM score between replicate spectra of the same molecule. To this end, we explicitly model the variation in instrument measurements of MS/MS peak intensities and show how these models can be used to determine a theoretical distribution of SSM scores between reference and query spectra of the same molecule. While the proposed spectral library generating function (SLGF) approach can be used to calculate theoretical distributions for any additive SSM score (e.g., any dot product), we further show how it can be used to calculate the distribution of expected cosines between reference and query spectra. We developed a spectral library search tool, Tremolo, and demonstrate that this SLGF-based search tool significantly outperforms current state-of-the-art spectral library search tools and provide a detailed discussion of the multiple reasons behind the observed differences in the sets of identified MS/MS spectra.

  17. Synthetic peptides.

    PubMed

    Francis, M J

    1996-01-01

    Efforts to produce more stable and defined vaccines have concentrated on studying, in detail, the immune response to many infectious diseases in order to identify the antigenic sites on the pathogens that are involved in stimulating protective immumty. Armed with this knowledge, it is possible to mimic such sites by producing short chains of amino acids (peptides) and to use these as the basis for novel vaccines. The earliest documented work on peptide immunization is actually for a plant virus, tobacco mosaic virus. In 1963, Anderer (1) demonstrated that rabbit antibodies to an isolated hexapeptide fragment from the virus-coat protein coupled to bovine serum albumm would neutralize the infectious vn-us in culture. Two years later, he used a synthetically produced copy of the same peptide to confirm this observation. This was pioneering work, and it was over 10 years before the next example of a peptide that elicited antivirus antibody appeared following work by Sela and his colleagues (2) on a virus, MS2 bacteriophage, which infects bacteria. The emergence of more accessible techniques for sequencing proteins in 1977, coupled with the ability to synthesize readily peptides already developed in 1963, heralded a decade of intensive research into experimental peptide vaccines. The first demonstration that peptides could elicit protective immunity in vivo, in addition to neutralizing activity in vitro, was obtained using a peptide from the VP1 coat protein of foot-and-mouth disease virus (FMDV) in 1982, with the guinea pig as a laboratory animal model (3, 4). PMID:21359696

  18. Collagen-like antimicrobial peptides.

    PubMed

    Masuda, Ryo; Kudo, Masakazu; Dazai, Yui; Mima, Takehiko; Koide, Takaki

    2016-11-01

    Combinatorial library composed of rigid rod-like peptides with a triple-helical scaffold was constructed. The component peptides were designed to have various combinations of basic and neutral (or hydrophobic) amino acid residues based on collagen-like (Gly-Pro-Yaa)-repeating sequences, inspired from the basic and amphiphilic nature of naturally occurring antimicrobial peptides. Screening of the peptide pools resulted in identification of antimicrobial peptides. A structure-activity relationship study revealed that the position of Arg-cluster at N-terminus and cystine knots at C-terminus in the triple helix significantly contributed to the antimicrobial activity. The most potent peptide RO-A showed activity against Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. In addition, Escherichia coli exposed to RO-A resulted in abnormal elongation of the cells. RO-A was also shown to have remarkable stability in human serum and low cytotoxicity to mammalian cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 453-459, 2016. PMID:27271210

  19. Engineering RNA phage MS2 virus-like particles for peptide display

    NASA Astrophysics Data System (ADS)

    Jordan, Sheldon Keith

    Phage display is a powerful and versatile technology that enables the selection of novel binding functions from large populations of randomly generated peptide sequences. Random sequences are genetically fused to a viral structural protein to produce complex peptide libraries. From a sufficiently complex library, phage bearing peptides with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified by infection of bacteria. For certain applications however, existing phage display platforms have limitations. One such area is in the field of vaccine development, where the goal is to identify relevant epitopes by affinity-selection against an antibody target, and then to utilize them as immunogens to elicit a desired antibody response. Today, affinity selection is usually conducted using display on filamentous phages like M13. This technology provides an efficient means for epitope identification, but, because filamentous phages do not display peptides in the high-density, multivalent arrays the immune system prefers to recognize, they generally make poor immunogens and are typically useless as vaccines. This makes it necessary to confer immunogenicity by conjugating synthetic versions of the peptides to more immunogenic carriers. Unfortunately, when introduced into these new structural environments, the epitopes often fail to elicit relevant antibody responses. Thus, it would be advantageous to combine the epitope selection and immunogen functions into a single platform where the structural constraints present during affinity selection can be preserved during immunization. This dissertation describes efforts to develop a peptide display system based on the virus-like particles (VLPs) of bacteriophage MS2. Phage display technologies rely on (1) the identification of a site in a viral structural protein that is

  20. PEP-on-DEP: A competitive peptide-based disposable electrochemical aptasensor for renin diagnostics.

    PubMed

    Biyani, Manish; Kawai, Keiko; Kitamura, Koichiro; Chikae, Miyuki; Biyani, Madhu; Ushijima, Hiromi; Tamiya, Eiichi; Yoneda, Takashi; Takamura, Yuzuru

    2016-10-15

    Antibody-based immunosensors are relatively less accessible to a wide variety of unreachable targets, such as low-molecular-weight biomarkers that represent a rich untapped source of disease-specific diagnostic information. Here, we present a peptide aptamer-based electrochemical sensor technology called 'PEP-on-DEP' to detect less accessible target molecules, such as renin, and to improve the quality of life. Peptide-based aptamers represent a relatively smart class of affinity binders and show great promise in biosensor development. Renin is involved in the regulation of arterial blood pressure and is an emerging biomarker protein for predicting cardiovascular risk and prognosis. To our knowledge, no studies have described aptamer molecules that can be used as new potent probes for renin. Here, we describe a portable electrochemical biosensor platform based on the newly identified peptide aptamer molecules for renin. We constructed a randomized octapeptide library pool with diversified sequences and selected renin specific peptide aptamers using cDNA display technology. We identified a few peptide aptamer sequences with a KD in the µM binding affinity range for renin. Next, we grafted the selected peptide aptamers onto gold nanoparticles and detected renin in a one-step competitive assay using our originally developed DEP (Disposable Electrochemical Printed) chip and a USB powered portable potentiostat system. We successfully detected renin in as little as 300ngmL(-1) using the PEP-on-DEP method. Thus, the generation and characterization of novel probes for unreachable target molecules by merging a newly identified peptide aptamer with electrochemical transduction allowed for the development of a more practical biosensor that, in principle, can be adapted to develop a portable, low-cost and mass-producible biosensor for point-of-care applications.

  1. Fluorogenic peptide sequences--transformation of short peptides into fluorophores under ambient photooxidative conditions.

    PubMed

    Juskowiak, Gary L; Stachel, Shawn J; Tivitmahaisoon, Parcharee; Van Vranken, David L

    2004-01-21

    Long-lived proteins are susceptible to nonenzymatic chemical reactions and the evolution of fluorescence; however, little is known about the sequence-dependence of fluorogenesis. We synthesized a library of over half a million octapeptides and exposed it to light and air in pH 7.4 buffer to identify fluorogenic peptides that evolve under mild oxidative conditions. The bead-based peptide library was composed of the general sequence H(2)N-Ala-(Xxx)(6)-Ala-resin, where Xxx was one of nine representative amino acids: Asp, Gly, His, Leu, Lys, Pro, Ser, Trp, and Tyr. Next, we selected five highly fluorescent beads from the library and subjected them to microsequencing, revealing the sequence of the unreacted peptide. All five of the fluorogenic sequences were ionic; lacked Tyr, His, and Leu; and most of the sequences contained only one Trp. We then synthesized the five soluble peptides corresponding to the fluorogenic peptide sequences and exposed them to photooxidative conditions. In general, the soluble peptides reacted slowly, generating nonfluorescent monooxygenated and dioxygenated products. However, one peptide (H(2)N-AlaLysProTrpGlyGlyAspAla-CONH(2)) evolved into a highly fluorescent photoproduct as well as a nonfluorescent monooxygenated photoproduct. The fluorescent photoproduct consisted of a 2-carboxy-quinolin-4-yl moiety fused to the N-terminus of GlyGlyAspAla. The formation of this photoproduct requires cleavage of the peptide backbone and a dramatic reorganization of tryptophan. This work demonstrates that sequencing unreacted peptide on beads can reveal sequences with unique nonenzymatic reactivity. The study also confirms that peptide fluorogenesis is dependent on sequence and not merely on the presence of tryptophan. The potential importance of fluorogenic peptide sequences is two-fold. First, fluorogenic sequences that arise through mutation could prove to be hot spots for human aging. Second, fluorogenic sequences, particularly those compatible with

  2. Entropy reduction in unfolded peptides (and proteins) due to conformational preferences of amino acid residues.

    PubMed

    Schweitzer-Stenner, Reinhard; Toal, Siobhan E

    2014-11-01

    As established by several groups over the last 20 years, amino acid residues in unfolded peptides and proteins do not exhibit the unspecific random distribution as assumed by the classical random coil model. Individual amino acid residues in small peptides were found to exhibit different conformational preferences. Here, we utilize recently obtained conformational distributions of guest amino acid residues in GxG peptides to estimate their conformational entropy, which we find to be significantly lower than the entropy of an assumed random coil like distribution. Only at high temperature do backbone entropies approach random coil like values. We utilized the obtained backbone entropies of the investigated amino acid residues to estimate the loss of conformational entropy caused by a coil → helix transition and identified two subsets of amino acid residues for which the thus calculated entropy losses correlate well with the respective Gibbs energy of helix formation obtained for alanine based host-guest systems. Calculated and experimentally derived entropic losses were found to be in good agreement. For most of the amino acid residues investigated entropic losses derived from our GxG distributions correlate very well with corresponding values recently obtained from MD simulations biased by conformational propensities derived from truncated coil libraries. Both, conformational entropy and the entropy of solvation exhibit a strong, residue specific temperature dependence, which can be expected to substantially affect the stability of unfolded states. Altogether, our results provide strong evidence for the notion that conformational preferences of amino acid residues matter with regard to the thermodynamics of peptide and protein folding.

  3. Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

    PubMed

    Phipps, M Lisa; Lillo, Antoinetta M; Shou, Yulin; Schmidt, Emily N; Paavola, Chad D; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I; Bradbury, Andrew R M; Martinez, Jennifer S

    2016-01-01

    Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands. PMID:27626637

  4. Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands

    PubMed Central

    Shou, Yulin; Schmidt, Emily N.; Paavola, Chad D.; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I.; Bradbury, Andrew R. M.; Martinez, Jennifer S.

    2016-01-01

    Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands. PMID:27626637

  5. Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

    PubMed

    Phipps, M Lisa; Lillo, Antoinetta M; Shou, Yulin; Schmidt, Emily N; Paavola, Chad D; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I; Bradbury, Andrew R M; Martinez, Jennifer S

    2016-01-01

    Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

  6. America's Star Libraries

    ERIC Educational Resources Information Center

    Lyons, Ray; Lance, Keith Curry

    2009-01-01

    "Library Journal"'s new national rating of public libraries, the "LJ" Index of Public Library Service, identifies 256 "star" libraries. It rates 7,115 public libraries. The top libraries in each group get five, four, or three Michelin guide-like stars. All included libraries, stars or not, can use their scores to learn from their peers and improve…

  7. University Libraries in Pakistan.

    ERIC Educational Resources Information Center

    Haider, Syed Jalaluddin

    1986-01-01

    This profile of university libraries in Pakistan covers history of higher education and role of the library; organization of library service (strong central library, decentralized library service, central library with department libraries); resources and collection building; technical processing; readers' services; administration and staffing;…

  8. Discovering and improving novel peptide therapeutics.

    PubMed

    McGregor, Duncan Patrick

    2008-10-01

    Peptides have a number of advantages over small molecules in terms of specificity and affinity for targets, and over antibodies in terms of size. However, sensitivity to serum and tissue proteases coupled with short serum half-life has resulted in few recombinant library derived peptides, making the transition from lead to drug on the market. Recently, a series of technologies have been developed to address both these issues: selection methodologies addressing protease resistance have been developed that when combined with methods such as pegylation antibody Fc attachment and binding to serum albumin look likely to finally turn therapeutic peptides into a widely accepted drug class.

  9. Peptide-Like Molecules (PLMs): A Journey from Peptide Bond Isosteres to Gramicidin S Mimetics and Mitochondrial Targeting Agents

    PubMed Central

    Wipf, Peter; Xiao, Jingbo; Stephenson, Corey R. J.

    2010-01-01

    Peptides are natural ligands and substrates for receptors and enzymes and exhibit broad physiological effects. However, their use as therapeutic agents often suffers from poor bioavailability and insufficient membrane permeability. The success of peptide mimicry hinges on the ability of bioisosteres, in particular peptide bond replacements, to adopt suitable secondary structures relevant to peptide strands and position functional groups in equivalent space. This perspective highlights past and ongoing studies in our group that involve new methods development as well as specific synthetic library preparations and applications in chemical biology, with the goal to enhance the use of alkene and cyclopropane peptide bond isosteres. PMID:20725595

  10. Selection of Novel Peptides Homing the 4T1 CELL Line: Exploring Alternative Targets for Triple Negative Breast Cancer

    PubMed Central

    Nobrega, Franklin L.; Martins, Ivone M.

    2016-01-01

    The use of bacteriophages to select novel ligands has been widely explored for cancer therapy. Their application is most warranted in cancer subtypes lacking knowledge on how to target the cancer cells in question, such as the triple negative breast cancer, eventually leading to the development of alternative nanomedicines for cancer therapeutics. Therefore, the following study aimed to select and characterize novel peptides for a triple negative breast cancer murine mammary carcinoma cell line– 4T1. Using phage display, 7 and 12 amino acid random peptide libraries were screened against the 4T1 cell line. A total of four rounds, plus a counter-selection round using the 3T3 murine fibroblast cell line, was performed. The enriched selective peptides were characterized and their binding capacity towards 4T1 tissue samples was confirmed by immunofluorescence and flow cytometry analysis. The selected peptides (4T1pep1 –CPTASNTSC and 4T1pep2—EVQSSKFPAHVS) were enriched over few rounds of selection and exhibited specific binding to the 4T1 cell line. Interestingly, affinity to the human MDA-MB-231 cell line was also observed for both peptides, promoting the translational application of these novel ligands between species. Additionally, bioinformatics analysis suggested that both peptides target human Mucin-16. This protein has been implicated in different types of cancer, as it is involved in many important cellular functions. This study strongly supports the need of finding alternative targeting systems for TNBC and the peptides herein selected exhibit promising future application as novel homing peptides for breast cancer therapy. PMID:27548261

  11. Selection of Novel Peptides Homing the 4T1 CELL Line: Exploring Alternative Targets for Triple Negative Breast Cancer.

    PubMed

    Silva, Vera L; Ferreira, Debora; Nobrega, Franklin L; Martins, Ivone M; Kluskens, Leon D; Rodrigues, Ligia R

    2016-01-01

    The use of bacteriophages to select novel ligands has been widely explored for cancer therapy. Their application is most warranted in cancer subtypes lacking knowledge on how to target the cancer cells in question, such as the triple negative breast cancer, eventually leading to the development of alternative nanomedicines for cancer therapeutics. Therefore, the following study aimed to select and characterize novel peptides for a triple negative breast cancer murine mammary carcinoma cell line- 4T1. Using phage display, 7 and 12 amino acid random peptide libraries were screened against the 4T1 cell line. A total of four rounds, plus a counter-selection round using the 3T3 murine fibroblast cell line, was performed. The enriched selective peptides were characterized and their binding capacity towards 4T1 tissue samples was confirmed by immunofluorescence and flow cytometry analysis. The selected peptides (4T1pep1 -CPTASNTSC and 4T1pep2-EVQSSKFPAHVS) were enriched over few rounds of selection and exhibited specific binding to the 4T1 cell line. Interestingly, affinity to the human MDA-MB-231 cell line was also observed for both peptides, promoting the translational application of these novel ligands between species. Additionally, bioinformatics analysis suggested that both peptides target human Mucin-16. This protein has been implicated in different types of cancer, as it is involved in many important cellular functions. This study strongly supports the need of finding alternative targeting systems for TNBC and the peptides herein selected exhibit promising future application as novel homing peptides for breast cancer therapy. PMID:27548261

  12. Library Venturing.

    ERIC Educational Resources Information Center

    Wilson, H. Donald

    1986-01-01

    There is opportunity for service and profit to imaginative libraries organizing to provide new forms of knowledge. Librarians as entrepreneurs must learn venture management and finance. Available assistance includes growing entrepreneural understanding in large institutions; family and friends; private wealth-seeking investment; new business…

  13. SPRNG Scalable Parallel Random Number Generator LIbrary

    2010-03-16

    This revision corrects some errors in SPRNG 1. Users of newer SPRNG versions can obtain the corrected files and build their version with it. This version also improves the scalability of some of the application-based tests in the SPRNG test suite. It also includes an interface to a parallel Mersenne Twister, so that if users install the Mersenne Twister, then they can test this generator with the SPRNG test suite and also use some SPRNGmore » features with that generator.« less

  14. SPRNG Scalable Parallel Random Number Generator LIbrary

    SciTech Connect

    Srinivasan, Ashok

    2010-03-16

    This revision corrects some errors in SPRNG 1. Users of newer SPRNG versions can obtain the corrected files and build their version with it. This version also improves the scalability of some of the application-based tests in the SPRNG test suite. It also includes an interface to a parallel Mersenne Twister, so that if users install the Mersenne Twister, then they can test this generator with the SPRNG test suite and also use some SPRNG features with that generator.

  15. Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

    PubMed

    Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

    2015-05-15

    Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

  16. Selection of high affinity peptide ligands for detection of circulating antibodies in neurocysticercosis.

    PubMed

    da Silva Ribeiro, Vanessa; Manhani, Marianna Nascimento; Cardoso, Rone; Vieira, Carlos Ueira; Goulart, Luiz Ricardo; Costa-Cruz, Julia Maria

    2010-04-01

    Neurocysticercosis (NC), caused by Taenia solium, is the most common infection caused by helminthes of the human central nervous system. In this study, a random peptide phage display library was used to isolate peptide ligands as potential markers for neurocysticercosis diagnosis, because occurrence of cross-reactions with other helminthes species in the current used markers. We selected different peptides using IgG purified from pooled sera of neurocysticercosis patients. To investigate the diagnostic potential of recombinant peptides, we have tested different panels of serum samples by Phage-ELISA, and 10 phage clones strongly bound to the anti-T. solium IgGs in NC sera, with an accuracy range from 84.2% to 95%. The phage clones, NC(4)1 and NC(2)8, presented the highest sensitivity and specificity (100%), respectively, and most important, some phage clones did not react with patients' sera from Echinococcus granulosus infected patients. The validation with a competitive ELISA assay demonstrated that the selected phages could mimic T. solium epitopes and bind specifically to the pool of NC sera. Finally, the two recombinant antigens may become potential biomarkers for serodiagnosis of NC, and the Phage-ELISA demonstrated to be a very good assay, being reproducible, simple, fast, and low-cost due to its production through Escherichia coli culture, allowing a high throughput screening of NC.

  17. Anti-heparan Sulfate Peptides That Block Herpes Simplex Virus Infection in Vivo*

    PubMed Central

    Tiwari, Vaibhav; Liu, Jian; Valyi-Nagy, Tibor; Shukla, Deepak

    2011-01-01

    Heparan sulfate (HS) and its highly modified form, 3-O-sulfated heparan sulfate (3-OS HS), contribute strongly to herpes simplex virus type-1 (HSV-1) infection in vitro. Here we report results from a random M13-phage display library screening to isolate 12-mer peptides that bind specifically to HS, 3-OS HS, and block HSV-1 entry. The screening identified representative candidates from two-different groups of anti-HS peptides with high positive charge densities. Group 1, represented by G1 peptide (LRSRTKIIRIRH), belongs to a class with alternating charges (XRXRXKXXRXRX), and group 2, represented by G2 peptide (MPRRRRIRRRQK), shows repetitive charges (XXRRRRXRRRXK). Viral entry and glycoprotein D binding assays together with fluorescent microscopy data indicated that both G1 and G2 were potent in blocking HSV-1 entry into primary cultures of human corneal fibroblasts and CHO-K1 cells transiently expressing different glycoprotein D receptors. Interestingly, G2 peptide isolated against 3-OS HS displayed wider ability to inhibit entry of clinically relevant strains of HSV-1 and some divergent members of herpesvirus family including cytomegalovirus and human herpesvirus-8. To identify functional residues within G1 and G2, we performed point mutations and alanine-scanning mutagenesis. Several arginine and a lysine residues were needed for anti-HSV-1 activity, suggesting the importance of the positively charged residues in virus-cell binding and virus-induced membrane fusion. In vivo administration of G1 or G2 peptide as a prophylactic eye drop completely blocked HSV-1 spread in the mouse cornea as evident by immunohistochemistry. This result also highlights an in vivo significance of HS and 3-OS HS during ocular herpes infection. PMID:21596749

  18. Art Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

    International Federation of Library Associations, The Hague (Netherlands).

    Papers on art libraries, librarianship, and documentation presented at the 1982 International Federation of Library Associations (IFLA) conference include: (1) "The Tyranny of Distance: Art Libraries in Canada," a description by Mary F. Williamson of Canada's regional art libraries which serve both art students and the general public; (2) "A…

  19. Antimicrobial peptides.

    PubMed

    Zhang, Ling-Juan; Gallo, Richard L

    2016-01-11

    Antimicrobial peptides and proteins (AMPs) are a diverse class of naturally occurring molecules that are produced as a first line of defense by all multicellular organisms. These proteins can have broad activity to directly kill bacteria, yeasts, fungi, viruses and even cancer cells. Insects and plants primarily deploy AMPs as an antibiotic to protect against potential pathogenic microbes, but microbes also produce AMPs to defend their environmental niche. In higher eukaryotic organisms, AMPs can also be referred to as 'host defense peptides', emphasizing their additional immunomodulatory activities. These activities are diverse, specific to the type of AMP, and include a variety of cytokine and growth factor-like effects that are relevant to normal immune homeostasis. In some instances, the inappropriate expression of AMPs can also induce autoimmune diseases, thus further highlighting the importance of understanding these molecules and their complex activities. This Primer will provide an update of our current understanding of AMPs. PMID:26766224

  20. Antimicrobial Peptides

    PubMed Central

    Bahar, Ali Adem; Ren, Dacheng

    2013-01-01

    The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics). PMID:24287494

  1. Antimicrobial peptides.

    PubMed

    Bahar, Ali Adem; Ren, Dacheng

    2013-11-28

    The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill "superbugs" emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics).

  2. 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

  3. Strong inhibition of beta-amyloid peptide aggregation realized by two-steps evolved peptides.

    PubMed

    Ghimire Gautam, Sunita; Komatsu, Masayuki; Nishigaki, Koichi

    2015-03-01

    Several decades of cumulated research evidence have proven that aggregation of beta-amyloid 42 (Aβ42) is the main cause of neuronal death in the brains of patients with Alzheimer's disease. Therefore, inhibition of Aβ42 aggregation holds great promise for the prevention and treatment of Alzheimer's disease. To this end, we used a systematic in vitro evolution including a paired peptide library method. We identified two peptides with high binding affinity (with Kd in the nm range) for Aβ42. Functionally, these peptides strongly inhibited the aggregation of Aβ42 as shown by the thioflavin T assay and atomic force microscopy. Moreover, these peptides rescued PC12 cells from the cytotoxic effect of aggregated Aβ42 in vitro. Our results suggest that these novel peptides may be potential therapeutic seeds for the treatment of Alzheimer's disease.

  4. Graph Library

    2007-06-12

    GraphLib is a support library used by other tools to create, manipulate, store, and export graphs. It provides a simple interface to specifS’ arbitrary directed and undirected graphs by adding nodes and edges. Each node and edge can be associated with a set of attributes describing size, color, and shape. Once created, graphs can be manipulated using a set of graph analysis algorithms, including merge, prune, and path coloring operations. GraphLib also has the abilitymore » to export graphs into various open formats such as DOT and GML.« less

  5. Cell Libraries

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A NASA contract led to the development of faster and more energy efficient semiconductor materials for digital integrated circuits. Gallium arsenide (GaAs) conducts electrons 4-6 times faster than silicon and uses less power at frequencies above 100-150 megahertz. However, the material is expensive, brittle, fragile and has lacked computer automated engineering tools to solve this problem. Systems & Processes Engineering Corporation (SPEC) developed a series of GaAs cell libraries for cell layout, design rule checking, logic synthesis, placement and routing, simulation and chip assembly. The system is marketed by Compare Design Automation.

  6. cis-Peptide Bonds: A Key for Intestinal Permeability of Peptides? .

    PubMed

    Marelli, Udaya Kiran; Ovadia, Oded; Frank, Andreas Oliver; Chatterjee, Jayanta; Gilon, Chaim; Hoffman, Amnon; Kessler, Horst

    2015-10-19

    Recent structural studies on libraries of cyclic hexapeptides led to the identification of common backbone conformations that may be instrumental to the oral availability of peptides. Furthermore, the observation of differential Caco-2 permeabilities of enantiomeric pairs of some of these peptides strongly supports the concept of conformational specificity driven uptake and also suggests a pivotal role of carrier-mediated pathways for peptide transport, especially for scaffolds of polar nature. This work presents investigations on the Caco-2 and PAMPA permeability profiles of 13 selected N-methylated cyclic pentaalanine peptides derived from the basic cyclo(-D-Ala-Ala4 -) template. These molecules generally showed moderate to low transport in intestinal epithelia with a few of them exhibiting a Caco-2 permeability equal to or slightly higher than that of mannitol, a marker for paracellular permeability. We identified that the majority of the permeable cyclic penta- and hexapeptides possess an N-methylated cis-peptide bond, a structural feature that is also present in the orally available peptides cyclosporine A and the tri-N-methylated analogue of the Veber-Hirschmann peptide. Based on these observations it appears that the presence of N-methylated cis-peptide bonds at certain locations may promote the intestinal permeability of peptides through a suitable conformational preorganization.

  7. Advances in synthetic peptides reagent discovery

    NASA Astrophysics Data System (ADS)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Stratis-Cullum, Dimitra N.

    2013-05-01

    Bacterial display technology offers a number of advantages over competing display technologies (e.g, phage) for the rapid discovery and development of peptides with interaction targeted to materials ranging from biological hazards through inorganic metals. We have previously shown that discovery of synthetic peptide reagents utilizing bacterial display technology is relatively simple and rapid to make laboratory automation possible. This included extensive study of the protective antigen system of Bacillus anthracis, including development of discovery, characterization, and computational biology capabilities for in-silico optimization. Although the benefits towards CBD goals are evident, the impact is far-reaching due to our ability to understand and harness peptide interactions that are ultimately extendable to the hybrid biomaterials of the future. In this paper, we describe advances in peptide discovery including, new target systems (e.g. non-biological materials), advanced library development and clone analysis including integrated reporting.

  8. Identifying proteins in zebrafish embryos using spectral libraries generated from dissected adult organs and tissues.

    PubMed

    van der Plas-Duivesteijn, Suzanne J; Mohammed, Yassene; Dalebout, Hans; Meijer, Annemarie; Botermans, Anouk; Hoogendijk, Jordy L; Henneman, Alex A; Deelder, André M; Spaink, Herman P; Palmblad, Magnus

    2014-03-01

    Spectral libraries provide a sensitive and accurate method for identifying peptides from tandem mass spectra, complementary to searching genome-derived databases or sequencing de novo. Their application requires comprehensive libraries including peptides from low-abundant proteins. Here we describe a method for constructing such libraries using biological differentiation to "fractionate" the proteome by harvesting adult organs and tissues and build comprehensive libraries for identifying proteins in zebrafish (Danio rerio) embryos and larvae (an important and widely used model system). Hierarchical clustering using direct comparison of spectra was used to prioritize organ selection. The resulting and publicly available library covers 14,164 proteins, significantly improved the number of peptide-spectrum matches in zebrafish developmental stages, and can be used on data from different instruments and laboratories. The library contains information on tissue and organ expression of these proteins and is also applicable for adult experiments. The approach itself is not limited to zebrafish but would work for any model system.

  9. Lithuanian Library History.

    ERIC Educational Resources Information Center

    Gudauskas, Renaldas

    1994-01-01

    Reviews the history of libraries in Lithuania from 1940 to 1992. Highlights include early book collections, and attitudes toward books and reading; changes after the Soviet invasion, including censorship, lack of timeliness, and information barriers; library networks; library education; research trends; library legislation; library literature; and…

  10. Rural Libraries in Illinois.

    ERIC Educational Resources Information Center

    Crossland, Brent, Ed.; And Others

    1993-01-01

    This theme issue includes 13 articles that discuss rural library concerns in Illinois. Topics addressed include strengthening library services; rural trends and their impact on libraries; partnerships; Cooperative Extension Service revitalization; financing; economic development; resource sharing in school libraries; and public libraries and user…

  11. Marketing the Virtual Library

    ERIC Educational Resources Information Center

    Fagan, Jody Condit

    2009-01-01

    Far more people are familiar with their local public or college library facility than their library's website and online resources. In fact, according to a recent survey, 96% of Americans said they had visited a library in person, but less than one-third have visited their online library. Since everyone agrees that online library resources are…

  12. Deutsches Museum Library.

    ERIC Educational Resources Information Center

    Berninger, Ernst H.; Reineke, Eva

    1986-01-01

    This history of the Library of the Deutsches Museum (Munich, Germany) covers the library's situation at end of the nineteenth century, library collections and buildings, use of the library for research, the rare book collection, and service statistics. Library director, collection and staff size, and main subjects collected are included. (EJS)

  13. Library Directions in 1988.

    ERIC Educational Resources Information Center

    DeCandido, GraceAnne A.

    1989-01-01

    Reviews major library issues and events of 1988, including: (1) the Federal Bureau of Investigation's Library Awareness Program; (2) cooperation with USSR libraries; (3) library finance; (4) preservation; and (5) special programing. News about a number of prominent library professionals is included in a sidebar. (MES)

  14. Rural Library Service.

    ERIC Educational Resources Information Center

    Vavrek, Bernard; And Others

    1989-01-01

    Five articles present an overview of trends and issues affecting rural libraries. The areas discussed include the status of rural library services; outreach programs; the role of library cooperation in the support of rural library service; the development of rural information centers; and political marketing of the rural library. (CLB)

  15. Allosterically Regulated Phosphatase Activity from Peptide-PNA Conjugates Folded Through Hybridization.

    PubMed

    Machida, Takuya; Dutt, Som; Winssinger, Nicolas

    2016-07-18

    The importance of spatial organization in short peptide catalysts is well recognized. We synthesized and screened a library of peptides flanked by peptide nucleic acids (PNAs) such that the peptide would be constrained in a hairpin loop upon hybridization. A screen for phosphatase activity led to the discovery of a catalyst with >25-fold rate acceleration over the linear peptide. We demonstrated that the hybridization-enforced folding of the peptide is necessary for activity, and designed a catalyst that is allosterically controlled using a complementary PNA sequence. PMID:27320214

  16. Affinity Selection of Peptide Binders with Magnetic Beads via Organic Phase Separation (MOPS).

    PubMed

    Murai, Ryuichi; Nogi, Taiki; Tateoka, Komei; Sato, Atsushi

    2015-01-01

    We describe a new method for affinity selection of peptide binders for soluble protein targets using magnetic beads via organic phase separation (MOPS) from a phage display library. As a model target molecule, a mouse monoclonal antibody against human integrin α9β1 (Y9A2) immobilized onto protein G magnetic beads was incubated with a 15-mer or 20-mer random peptide phage-display library. The suspensions containing the phage-magnetic beads conjugates were then transferred onto the organic phase and centrifuged in order to recover the Y9A2 bound phage immobilized on the protein G magnetic beads in the lower organic phase. After three rounds of biopanning, we were able to isolate specific phage clones that could not be obtained by the conventional approach. Furthermore, this new approach was found to be highly effective for isolating phage-binders for Fc-fusion constructs; indeed, enrichment of specific phage-binders was observed after only the first panning cycle. Thus, MOPS can improve the selection of specific phage-binders for soluble protein targets mainly due to the removal of non-specific binders.

  17. The mzqLibrary--An open source Java library supporting the HUPO-PSI quantitative proteomics standard.

    PubMed

    Qi, Da; Zhang, Huaizhong; Fan, Jun; Perkins, Simon; Pisconti, Addolorata; Simpson, Deborah M; Bessant, Conrad; Hubbard, Simon; Jones, Andrew R

    2015-09-01

    The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML-based format, capable of representing data about two-dimensional features from LC-MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java-based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer. The mzqLibrary contains routines for mapping (peptide) identifications on quantified features, inference of protein (group)-level quantification values from peptide-level values, normalisation and basic statistics for differential expression. These routines can be accessed via the command line, via a Java programming interface access or a basic graphical user interface. The mzqLibrary also contains several file format converters, including import converters (to mzQuantML) from OpenMS, Progenesis LC-MS and MaxQuant, and exporters (from mzQuantML) to other standards or useful formats (mzTab, HTML, csv). The mzqViewer contains in-built routines for viewing the tables of data (about features, peptides or proteins), and connects to the R statistical library for more advanced plotting options. The mzqLibrary and mzqViewer packages are available from https://code.google.com/p/mzq-lib/.

  18. Porcine parvovirus removal using trimer and biased hexamer peptides

    PubMed Central

    Heldt, Caryn L.; Gurgel, Patrick V.; Jaykus, Lee-Ann; Carbonell, Ruben G.

    2014-01-01

    Assuring the microbiological safety of biological therapeutics remains an important concern. Our group has recently reported small trimeric peptides that have the ability to bind and remove a model non-enveloped virus, porcine parvovirus (PPV), from complex solutions containing human blood plasma. In an effort to improve the removal efficiency of these small peptides, we created a biased library of hexamer peptides that contain two previously reported trimeric peptides designated WRW and KYY. This library was screened and several hexamer peptides were discovered that also removed PPV from solution, but there was no marked improvement in removal efficiency when compared to the trimeric peptides. Based on simulated docking experiments, it appeared that hexamer peptide binding is dictated more by secondary structure, whereas the binding of trimeric peptides is dominated by charge and hydrophobicity. This study demonstrates that trimeric and hexameric peptides may have different, matrix-specific roles to play in virus removal applications. In general, the hexamer ligand may perform better for binding of specific viruses, whereas the trimer ligand may have more broadly reactive virus-binding properties. PMID:21751387

  19. The Whole Library Handbook 3: Current Data, Professional Advice, and Curiosa about Libraries and Library Services.

    ERIC Educational Resources Information Center

    Eberhart, George M., Comp.

    This handbook contains articles, guidelines, and other information from the field of library science organized into the following chapters: (1) "Libraries," including some basic figures, academic libraries, public libraries, school libraries, special libraries, national libraries, state libraries, small libraries, facilities, the past, and the…

  20. C-Peptide Test

    MedlinePlus

    ... C-peptide is a useful marker of insulin production. The following are some purposes of C-peptide ... it nearly impossible to directly evaluate endogenous insulin production. In these cases, C-peptide measurement is a ...

  1. Agricultural Libraries and Information.

    ERIC Educational Resources Information Center

    Russell, Keith W., Ed.; Pisa, Maria G., Ed.

    1990-01-01

    Eleven articles address issues relating to agricultural libraries and information, including background on agricultural libraries and information, trend management, document delivery, reference services, user needs and library services, collection development, technologies for international information management, information sources,…

  2. Codon randomization for rapid exploration of chemical space in thiopeptide antibiotic variants

    PubMed Central

    Young, Travis S; Dorrestein, Pieter C; Walsh, Christopher T

    2012-01-01

    SUMMARY Thiopeptide antibiotics exhibit a profound level of chemical diversity that is installed through cascades of posttranslational modifications on ribosomal peptides. Here we present a technique to rapidly explore the chemical space of the thiopeptide GE37468 through codon randomization, yielding insights into thiopeptide maturation as well as structure and activity relationships. In this incarnation of the methodology, we randomized 7 residues of the prepeptide coding region, enabling the generation of 133 potential thiopeptide variants. Variant libraries were subsequently queried in two ways. First, high through-put MALDI-TOF mass spectrometry was applied to colony-level expressions to sample mutants which permitted full maturation of the antibiotic. Second, the activity of producing mutants was detected in an antibiotic overlay assay. In total, 29 of the 133 variants were found to produce mature compound, 12 of which retained antibiotic activity and one which had improved activity against Methicillin-resistant Staphylococcus aureus (MRSA). PMID:23261603

  3. Linking genotype to phenotype on beads: high throughput selection of peptides with biological function.

    PubMed

    Huang, Li-Chieh; Pan, Xiaoyan; Yang, Hongbing; Wan, Lai Kin Derek; Stewart-Jones, Guillaume; Dorrell, Lucy; Ogg, Graham

    2013-10-23

    Although peptides are well recognised biological molecules in vivo, their selection from libraries is challenging because of relative low affinity whilst in linear conformation. We hypothesized that multiplexed peptides and DNA on the surface of beads would provide a platform for enhanced avidity and the selection of relevant peptides from a library (ORBIT bead display). Using human immunodeficiency virus (HIV-1) gp120 as a target, we identify peptides that inhibit HIV-1 replication in vitro through blocking of protein:protein interaction with the co-receptor CCR5. The bead display approach has many potential applications for probing biological systems and for drug lead development.

  4. 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

  5. Alabama Public Library Service Library Directory and 1996 Statistical Report.

    ERIC Educational Resources Information Center

    Alabama Public Library Service, Montgomery.

    This publication presents library contact information and statistics for Alabama public libraries for fiscal year 1996 (October 1, 1995-September 30, 1996). The library directory is arranged by type of library: public libraries, single-county public library systems, multi-county public library systems, and multitype library systems. Entries…

  6. The Library of Virginia's Digital Library Project.

    ERIC Educational Resources Information Center

    Roderick, Elizabeth; Taylor, Jean Marie; Byrd, Sam; Courson, Glenn

    1997-01-01

    Describes The Library of Virginia's Digital Library Project that has made many of its state library collections available via the Internet and World Wide Web. Highlights include digitization decisions; the HTML Web gateway; the online catalog; microfilm digitization; users and use statistics; and future projects. (LRW)

  7. [Ants: a chemical library of anticancer molecules].

    PubMed

    Vétillard, Angélique; Bouzid, Wafa

    2016-01-01

    Animal venoms are complex mixtures containing simple organic molecules, proteins, peptides, and other bioactive elements with extraordinary biological properties associated with their ability to act on a number of molecular receptors in the process of incapacitating their target organisms. In such a context, arthropod venoms are invaluable sources of bioactive substances, with therapeutic interest but the limited availability of some venom such as those from ants, has restricted the potential that these biomolecules could represent. We investigated for the first time transcriptomic expression from the ant species Tetramorium bicarinatum. Four hundred randomly selected clones from cDNA libraries were sequenced and a total of 374 expressed sequence tags (ESTs) were generated. Based on the results of BLAST searches, these sequences were clustered and assembled into 269 contigs. About 72% (269) of these matched BLASTx hits with an interesting diversity and unusual abundance of cellular transcripts (48%) related to gene and protein expression reflecting the specialization of this tissue. In addition, transcripts encoding transposases were relatively highly expressed (14%). It may be that transposable elements are present and that their presence accounts for some of the variation in venom toxins. About twenty per cent of the ESTs were categorized as putative toxins, the major part represented by allergens (48% of the total venom toxins) such as pilosulin 5, sol i 3 and Myp p I and II. Several contigs encoding enzymes, including zinc-metalloproteases (17%) that are likely involved in the processing and activation of venom proteins/peptides, were also identified from the library. In addition, a number of sequences (8%) had no significant similarity to any known sequence which indicates a potential source of for the discovery of new toxins. In order to provide a global insight on the transcripts expressed in the venom gland of the Brazilian ant species Tetramorium

  8. [Ants: a chemical library of anticancer molecules].

    PubMed

    Vétillard, Angélique; Bouzid, Wafa

    2016-01-01

    Animal venoms are complex mixtures containing simple organic molecules, proteins, peptides, and other bioactive elements with extraordinary biological properties associated with their ability to act on a number of molecular receptors in the process of incapacitating their target organisms. In such a context, arthropod venoms are invaluable sources of bioactive substances, with therapeutic interest but the limited availability of some venom such as those from ants, has restricted the potential that these biomolecules could represent. We investigated for the first time transcriptomic expression from the ant species Tetramorium bicarinatum. Four hundred randomly selected clones from cDNA libraries were sequenced and a total of 374 expressed sequence tags (ESTs) were generated. Based on the results of BLAST searches, these sequences were clustered and assembled into 269 contigs. About 72% (269) of these matched BLASTx hits with an interesting diversity and unusual abundance of cellular transcripts (48%) related to gene and protein expression reflecting the specialization of this tissue. In addition, transcripts encoding transposases were relatively highly expressed (14%). It may be that transposable elements are present and that their presence accounts for some of the variation in venom toxins. About twenty per cent of the ESTs were categorized as putative toxins, the major part represented by allergens (48% of the total venom toxins) such as pilosulin 5, sol i 3 and Myp p I and II. Several contigs encoding enzymes, including zinc-metalloproteases (17%) that are likely involved in the processing and activation of venom proteins/peptides, were also identified from the library. In addition, a number of sequences (8%) had no significant similarity to any known sequence which indicates a potential source of for the discovery of new toxins. In order to provide a global insight on the transcripts expressed in the venom gland of the Brazilian ant species Tetramorium

  9. Dietary peptides improve wound healing following surgery.

    PubMed

    Roberts, P R; Black, K W; Santamauro, J T; Zaloga, G P

    1998-03-01

    To determine if peptide-based enteral diets improve wound healing when compared to amino acid-based diets, a prospective randomized study was conducted using 38 male Sprague-Dawley rats. Following placement of a standardized abdominal wound, 20 animals were randomized to an isonitrogenous peptide-based (PEP) versus amino acid-based diet (AA) for 10 d. In addition, 18 animals were randomized to an amino acid-based diet supplemented with the peptide carnosine (CARN) or its constituent amino acids (Control). Diets were administered through small bowel feeding tubes. Wound bursting pressure was significantly higher in the PEP animals compared to the AA animals (179+/-9 versus 138+/-12 mmHg; P=0.02). In addition, wound bursting pressure was significantly greater in the CARN animals compared to the Control animals (143+/-10 versus 116+/-8 mmHg; P=0.005). Peptide-based enteral diets improve wound healing when compared to nonpeptide generating amino acid-based diets. We also conclude that the dietary peptide carnosine represents a dietary peptide that improves wound healing when administered as part of a complete enteral formula. This effect on wound healing may be clinically relevant because carnosine is not found in most enteral formulas.

  10. Large scale production of phage antibody libraries using a bioreactor.

    PubMed

    Ferrara, Fortunato; Kim, Chang-Yub; Naranjo, Leslie A; Bradbury, Andrew R M

    2015-01-01

    One of the limitations of the use of phage antibody libraries in high throughput selections is the production of sufficient phage antibody library at the appropriate quality. Here, we successfully adapt a bioreactor-based protocol for the production of phage peptide libraries to the production of phage antibody libraries. The titers obtained in the stirred-tank bioreactor are 4 to 5 times higher than in a standard shake flask procedure, and the quality of the phage antibody library produced is indistinguishable to that produced using standard procedures as assessed by Western blotting and functional selections. Availability of this protocol will facilitate the use of phage antibody libraries in high-throughput scale selections.

  11. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested.

  12. Prison Libraries Inside Out.

    ERIC Educational Resources Information Center

    Singer, Glen

    2000-01-01

    Discussion of prison libraries provides an inside look at the correctional institution environment, prison security concerns, inmate patrons and library use, library collections and services, librarians and staff, the day-to-day operation of a prison library, and future possibilities and needs. (Author/LRW)

  13. Inside Prison Libraries.

    ERIC Educational Resources Information Center

    Vogel, Brenda; And Others

    1989-01-01

    Issues related to prison libraries are discussed in six articles. Topics covered include the history of American penitentiary ideology; standards for prison libraries; the controversy as to whether prison libraries should serve prisoners or be used as penological tools; and the lack of knowledge about prison libraries within the general library…

  14. Libraries, Ebooks, and Competition

    ERIC Educational Resources Information Center

    Hellman, Eric

    2010-01-01

    People keep writing articles about how valuable libraries are, even with ebooks and the Internet. What people are overlooking is that the reason libraries are having such fits dealing with a changing environment is not that libraries are unrecognized as fountains of value, it's that libraries are so valuable that they attract voracious new…

  15. Alaska Library Directory, 1996.

    ERIC Educational Resources Information Center

    Jennings, Mary, Ed.

    This directory of Alaska's Libraries lists: members of the Alaska Library Association (AkLA) Executive Council and Committee Chairs; State Board of Education members; members of the Governor's Advisory Council on Libraries; school, academic and public libraries and their addresses, phone and fax numbers, and contact persons; personal,…

  16. School Libraries in Hawaii.

    ERIC Educational Resources Information Center

    Bard, Therese Bissen

    This paper outlines the history, functions, administration, and current focus of school library services in Hawaii, which is the only state in the United States with a library staffed by a trained librarian in every public school. Its first school library was established in 1882. Elementary school libraries developed concurrently with secondary…

  17. California: Library Information Technologies.

    ERIC Educational Resources Information Center

    Will, Barbara, Ed.

    1996-01-01

    Describes six information technology projects in California libraries, including Internet access in public libraries; digital library developments at the University of California, Berkeley; the World Wide Web home page for the state library; Pacific Bell's role in statewide connectivity; state government initiatives; and services of the state…

  18. College and University Libraries.

    ERIC Educational Resources Information Center

    Shubert, Joseph F., Ed.; Josey, E. J., Ed.

    1986-01-01

    Following an introductory discussion by E. J. Josey that provides a perspective on college and university libraries, the following essays are presented: (1) "Academic Library Planning--Definitions and Early Planning Studies in Academic Libraries" (Stanton F. Biddle); (2) "Academic Libraries and Academic Computing--Rationale for a Modern Marriage"…

  19. AMERICA 2000 Library Partnership.

    ERIC Educational Resources Information Center

    Office of Educational Research and Improvement (ED), Washington, DC.

    The United States Department of Education, the National Endowment for the Humanities, the Library of Congress, the National Commission on Libraries and Information Science, and the National Institute for Literacy have formed the AMERICA 2000 Library Partnership to support libraries in their work toward the six National Education Goals announced by…

  20. Conservation of Library Materials.

    ERIC Educational Resources Information Center

    Illinois Libraries, 1985

    1985-01-01

    Twelve articles cover books as artifacts; workstations for conservation of library materials; care of scrapbooks, albums, and photographs; map preservation; library environment; flood recovery; disaster prevention and preparedness; incorporating preservation into library organization; and bibliography of Chester Public Library (Illinois) First…

  1. Special Library Services.

    ERIC Educational Resources Information Center

    Ensley, Robert F., Ed.

    1975-01-01

    The September 1975 issue of Illinois Libraries focuses on the needs of the developmentally disabled, physically handicapped, and emotionally disturbed. Articles on library services to the blind and physically handicapped cover standards, services of local public libraries, Library of Congress programs, braille books and sound recordings,…

  2. Growing Competition for Libraries.

    ERIC Educational Resources Information Center

    Gibbons, Susan

    2001-01-01

    Describes the Questia subscription-based online academic digital books library. Highlights include weaknesses of the collection; what college students want from a library; importance of marketing; competition for traditional academic libraries that may help improve library services; and the ability of Questia to overcome barriers and…

  3. Economics of Academic Libraries.

    ERIC Educational Resources Information Center

    Baumol, William J.; Marcus, Matityahu

    An analysis is conducted of economic issues pertinent to library planning in higher education in the face of rising costs and diminishing financial support. The individual chapters deal with: 1) growth rates in large university libraries; 2) library costs in colleges and universities; 3) cost trends and long-range plans; 4) library data; and 5) a…

  4. Facility Focus: Libraries.

    ERIC Educational Resources Information Center

    College Planning & Management, 2002

    2002-01-01

    Describes the design of the Charles V. Park Library at Central Michigan University and the Martha Rivers and E. Bronson Ingram Library, an addition to the Frederick Ferris Thompson Memorial Library at Vassar College. Discusses the libraries as examples of merging tradition with technology. Includes photographs. (EV)

  5. Idaho Library Trustee Manual.

    ERIC Educational Resources Information Center

    Idaho State Library, Boise.

    The public library board of trustees is an example of the great democratic tradition of giving authority for the operation of public agencies to members of the general public. The public library board is legally responsible for overseeing the efficient and effective operation of the public library. The board hires the library's administrator,…

  6. Selection of peptide inhibitors of interactions involved in complex protein assemblies: association of the core and surface antigens of hepatitis B virus.

    PubMed Central

    Dyson, M R; Murray, K

    1995-01-01

    As an example for studies of contacts involved in complex biological systems, peptide ligands that bind to the core antigen of hepatitis B virus (HBcAg) have been selected from a random hexapeptide library displayed on filamentous phage. Affinity-purified phage bearing aa sequence LLGRMK, or some related sequences, bound full-length or truncated HBcAg but did not bind denatured HBcAg. The long (L), but not the short (S), hepatitis B virus envelope polypeptide, when synthesized in an in vitro system, bound firmly to HBcAg, indicating that interaction between HBcAg and the pre-S region of the L polypeptide is critical for virus morphogenesis. This interaction was inhibited by peptide ALLGRMKG, suggesting that this and related small molecules may inhibit viral assembly. Images Fig. 2 PMID:7892246

  7. Proof of concept study with an HER-2 mimotope anticancer vaccine deduced from a novel AAV-mimotope library platform

    PubMed Central

    Singer, Josef; Manzano-Szalai, Krisztina; Fazekas, Judit; Thell, Kathrin; Bentley-Lukschal, Anna; Stremnitzer, Caroline; Roth-Walter, Franziska; Weghofer, Margit; Ritter, Mirko; Pino Tossi, Kerstin; Hörer, Markus; Michaelis, Uwe; Jensen-Jarolim, Erika

    2016-01-01

    ABSTRACT Background: Anticancer vaccines could represent a valuable complementary strategy to established therapies, especially in settings of early stage and minimal residual disease. HER-2 is an important target for immunotherapy and addressed by the monoclonal antibody trastuzumab. We have previously generated HER-2 mimotope peptides from phage display libraries. The synthesized peptides were coupled to carriers and applied for epitope-specific induction of trastuzumab-like IgG. For simplification and to avoid methodological limitations of synthesis and coupling chemistry, we herewith present a novel and optimized approach by using adeno-associated viruses (AAV) as effective and high-density mimotope-display system, which can be directly used for vaccination. Methods: An AAV capsid display library was constructed by genetically incorporating random peptides in a plasmid encoding the wild-type AAV2 capsid protein. AAV clones, expressing peptides specifically reactive to trastuzumab, were employed to immunize BALB/c mice. Antibody titers against human HER-2 were determined, and the isotype composition and functional properties of these were tested. Finally, prophylactically immunized mice were challenged with human HER-2 transfected mouse D2F2/E2 cells. Results: HER-2 mimotope AAV-vaccines induced antibodies specific to human HER-2. Two clones were selected for immunization of mice, which were subsequently grafted D2F2/E2 cells. Both mimotope AAV clones delayed the growth of tumors significantly, as compared to controls. Conclusion: In this study, a novel mimotope AAV-based platform was created allowing the isolation of mimotopes, which can be directly used as anticancer vaccines. The example of trastuzumab AAV-mimotopes demonstrates that this vaccine strategy could help to establish active immunotherapy for breast-cancer patients. PMID:27622022

  8. Proof of concept study with an HER-2 mimotope anticancer vaccine deduced from a novel AAV-mimotope library platform

    PubMed Central

    Singer, Josef; Manzano-Szalai, Krisztina; Fazekas, Judit; Thell, Kathrin; Bentley-Lukschal, Anna; Stremnitzer, Caroline; Roth-Walter, Franziska; Weghofer, Margit; Ritter, Mirko; Pino Tossi, Kerstin; Hörer, Markus; Michaelis, Uwe; Jensen-Jarolim, Erika

    2016-01-01

    ABSTRACT Background: Anticancer vaccines could represent a valuable complementary strategy to established therapies, especially in settings of early stage and minimal residual disease. HER-2 is an important target for immunotherapy and addressed by the monoclonal antibody trastuzumab. We have previously generated HER-2 mimotope peptides from phage display libraries. The synthesized peptides were coupled to carriers and applied for epitope-specific induction of trastuzumab-like IgG. For simplification and to avoid methodological limitations of synthesis and coupling chemistry, we herewith present a novel and optimized approach by using adeno-associated viruses (AAV) as effective and high-density mimotope-display system, which can be directly used for vaccination. Methods: An AAV capsid display library was constructed by genetically incorporating random peptides in a plasmid encoding the wild-type AAV2 capsid protein. AAV clones, expressing peptides specifically reactive to trastuzumab, were employed to immunize BALB/c mice. Antibody titers against human HER-2 were determined, and the isotype composition and functional properties of these were tested. Finally, prophylactically immunized mice were challenged with human HER-2 transfected mouse D2F2/E2 cells. Results: HER-2 mimotope AAV-vaccines induced antibodies specific to human HER-2. Two clones were selected for immunization of mice, which were subsequently grafted D2F2/E2 cells. Both mimotope AAV clones delayed the growth of tumors significantly, as compared to controls. Conclusion: In this study, a novel mimotope AAV-based platform was created allowing the isolation of mimotopes, which can be directly used as anticancer vaccines. The example of trastuzumab AAV-mimotopes demonstrates that this vaccine strategy could help to establish active immunotherapy for breast-cancer patients.

  9. Recursive deconvolution of combinatorial chemical libraries.

    PubMed

    Erb, E; Janda, K D; Brenner, S

    1994-11-22

    A recursive strategy that solves for the active members of a chemical library is presented. A pentapeptide library with an alphabet of Gly, Leu, Phe, and Tyr (1024 members) was constructed on a solid support by the method of split synthesis. One member of this library (NH2-Tyr-Gly-Gly-Phe-Leu) is a native binder to a beta-endorphin antibody. A variation of the split synthesis approach is used to build the combinatorial library. In four vials, a member of the library's alphabet is coupled to a solid support. After each coupling, a portion of the resin from each of the four reaction vials was set aside and catalogued. The solid support from each vial is then combined, mixed, and redivided. The steps of (i) coupling, (ii) saving and cataloging, and (iii) randomizing were repeated until a pentapeptide library was obtained. The four pentapeptide libraries where the N-terminal amino acid is defined were screened against the beta-endorphin antibody and quantitated via an ELISA. The amino acid of the four pools that demonstrated the most binding was then coupled to the four tetrapeptide partial libraries that had been set aside and catalogued during the split synthesis. This recursive deconvolution was repeated until the best binders were deduced. Besides the anticipated native binder, two other members of the library displayed significant binding. This recursive method of deconvolution does not use a molecular tag, requires only one split synthesis, and can be applied to the deconvolution of nonlinear small-molecule combinatorial libraries and linear oligomeric combinatorial libraries, since it is based only on the procedure of the synthesis. PMID:7972077

  10. Beyond helper phage: Using "helper cells" to select peptide affinity ligands

    DOE PAGESBeta

    Phipps, Mary Lisa; Lillo, Antoinetta M.; Shou, Yulin; Schmidt, Emily N.; Paavola, Chad D.; Naranjo, Leslie A.; Bemdich, Sara; Swanson, Basil I.; Bradbury, Andrew R. M.; Martinez, Jennifer S.; et al

    2016-09-14

    Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report onmore » the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Here, based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.« less

  11. 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.

  12. America's Star Libraries, 2010: Top-Rated Libraries

    ERIC Educational Resources Information Center

    Lyons, Ray; Lance, Keith Curry

    2010-01-01

    The "LJ" Index of Public Library Service 2010, "Library Journal"'s national rating of public libraries, identifies 258 "star" libraries. Created by Ray Lyons and Keith Curry Lance, and based on 2008 data from the IMLS, it rates 7,407 public libraries. The top libraries in each group get five, four, or three stars. All included libraries, stars or…

  13. Adding energy minimization strategy to peptide-design algorithm enables better search for RNA-binding peptides: Redesigned λ N peptide binds boxB RNA.

    PubMed

    Xiao, Xingqing; Hung, Michelle E; Leonard, Joshua N; Hall, Carol K

    2016-10-15

    Our previously developed peptide-design algorithm was improved by adding an energy minimization strategy which allows the amino acid sidechains to move in a broad configuration space during sequence evolution. In this work, the new algorithm was used to generate a library of 21-mer peptides which could substitute for λ N peptide in binding to boxB RNA. Six potential peptides were obtained from the algorithm, all of which exhibited good binding capability with boxB RNA. Atomistic molecular dynamics simulations were then conducted to examine the ability of the λ N peptide and three best evolved peptides, viz. Pept01, Pept26, and Pept28, to bind to boxB RNA. Simulation results demonstrated that our evolved peptides are better at binding to boxB RNA than the λ N peptide. Sequence searches using the old (without energy minimization strategy) and new (with energy minimization strategy) algorithms confirm that the new algorithm is more effective at finding good RNA-binding peptides than the old algorithm. © 2016 Wiley Periodicals, Inc.

  14. Exploration of structure-activity relationships at the two C-terminal residues of potent 11mer Glucagon-Like Peptide-1 receptor agonist peptides via parallel synthesis.

    PubMed

    Haque, Tasir S; Martinez, Rogelio L; Lee, Ving G; Riexinger, Douglas G; Lei, Ming; Feng, Ming; Koplowitz, Barry; Mapelli, Claudio; Cooper, Christopher B; Zhang, Ge; Huang, Christine; Ewing, William R; Krupinski, John

    2010-07-01

    We report the identification of potent agonists of the Glucagon-Like Peptide-1 receptor (GLP-1R) via evaluation of two positional scanning libraries and a two-dimensional focused library, synthesized in part on SynPhase Lanterns. These compounds are 11 amino acid peptides containing several unnatural amino acids, including (in particular) analogs of biphenylalanine (Bip) at the two C-terminal positions. Typical activities of the most potent peptides in this class are in the picomolar range in an in vitro functional assay using human GLP-1 receptor.

  15. Phage Display Selection of Cyclic Peptides That Inhibit Andes Virus Infection▿

    PubMed Central

    Hall, Pamela R.; Hjelle, Brian; Njus, Hadya; Ye, Chunyan; Bondu-Hawkins, Virginie; Brown, David C.; Kilpatrick, Kathleen A.; Larson, Richard S.

    2009-01-01

    Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents. PMID:19515773

  16. Hydroxyapatite-binding peptides for bone growth and inhibition

    DOEpatents

    Bertozzi, Carolyn R.; Song, Jie; Lee, Seung-Wuk

    2011-09-20

    Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

  17. Status of Higher Secondary School Libraries in Thiruvallur District

    ERIC Educational Resources Information Center

    Seenivasan, M.; Kumar, N. Ashok

    2014-01-01

    Libraries are the main sources of knowledge. They play a major role in fostering reading habit among school children. Hence, it is deemed interactive to study the status of higher secondary school libraries in Thiruvallur District, Tamil Nadu. For the Analysis 50 Higher Secondary Schools were selected randomly comprising of Government Higher…

  18. Summer library reading programs.

    PubMed

    Fiore, Carole D

    2007-01-01

    Virtually all public libraries in the United States provide some type of summer library reading program during the traditional summer vacation period. Summer library reading programs provide opportunities for students of many ages and abilities to practice their reading skills and maintain skills that are developed during the school year. Fiore summarizes some of the research in the field and relates it to library programs and usage by students. Several traditional and innovative programs from U.S. and Canadian libraries are described. She concludes with a call for further research related to summer library reading programs.

  19. Simulation of Peptides at Aqueous Interfaces

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Wilson, M.; Chipot, C.; DeVincenzi, Donald L. (Technical Monitor)

    2001-01-01

    Behavior of peptides at water-membrane interfaces is of great interest in studies on cellular transport and signaling, membrane fusion, and the action of toxins and antibiotics. Many peptides, which exist in water only as random coils, can form sequence-dependent, ordered structures at aqueous interfaces, incorporate into membranes and self-assembly into functional units, such as simple ion channels. Multi -nanosecond molecular dynamics simulations have been carried out to study the mechanism and energetics of interfacial folding of both non-polar and amphiphilic peptides, their insertion into membranes and association into higher-order structures. The simulations indicate that peptides fold non-sequentially, often through a series of amphiphilic intermediates. They further incorporate into the membrane in a preferred direction as folded monomers, and only then aggregate into dimers and, possibly, further into "dimers of dimers".

  20. Self-made phage libraries with heterologous inserts in the Mtd of Bordetella bronchiseptica.

    PubMed

    Overstreet, Cathie M; Yuan, Tom Z; Levin, Aron M; Kong, Calvin; Coroneus, John G; Weiss, Gregory A

    2012-04-01

    Phage display libraries are widely used as tools for identifying, dissecting and optimizing ligands. Development of a simple method to access greater library diversities could expedite and expand the technique. This paper reports progress toward harnessing the naturally occurring diversity generating retroelement used by Bordetella bronchiseptica bacteriophage to alter its tail-fiber protein. Mutagenesis and testing identified four sites amenable to the insertion of <19-residue heterologous peptides within the variable region. Such sites allow auto-generation of peptide libraries surrounded by a scaffold with additional variations. The resultant self-made phage libraries were used successfully for selections targeting anti-FLAG antibody, immobilized metal affinity chromatography microtiter plates and HIV-1 gp41. The reported experiments demonstrate the utility of the major tropism determinant protein of B.bronchiseptica as a natural scaffold for diverse, phage-constructed libraries with heterologous self-made phage libraries.

  1. One-step surface modification of polyurethane using affinity binding peptides for enhanced fouling resistance.

    PubMed

    Wang, Yibing; Yu, Yong; Zhang, Liting; Qin, Peng; Wang, Ping

    2015-01-01

    Affinity binding peptides were examined for surface fabrication of synthetic polymeric materials. Peptides possessing strong binding affinities toward polyurethane (PU) were discovered via biopanning of M13 phage peptide library. The apparent binding constant (K(app)) was as high as 2.68 × 10(9) M(-1) with surface peptide density exceeded 1.8 μg/cm(2). Structural analysis showed that the ideal peptide had a high content (75%) of H-donor amino acid residues, and that intensified hydrogen bond interaction was the key driving force for the highly stable binding of peptides on PU. PU treated with such affinity peptides promises applications as low-fouling materials, as peptides increased its wettability and substantially reduced protein adsorption and cell adhesion. These results demonstrated a facile but highly efficient one-step strategy for surface property modification of polymeric materials for biotechnological applications. PMID:25732121

  2. Deep sequencing analysis of phage libraries using Illumina platform.

    PubMed

    Matochko, Wadim L; Chu, Kiki; Jin, Bingjie; Lee, Sam W; Whitesides, George M; Derda, Ratmir

    2012-09-01

    This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10(7) reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2×10(7) reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6bp), one complimentary region (12bp) and a variable region (36bp). We applied this sequencing to a model library of 10(6) unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve

  3. SuperMimic – Fitting peptide mimetics into protein structures

    PubMed Central

    Goede, Andrean; Michalsky, Elke; Schmidt, Ulrike; Preissner, Robert

    2006-01-01

    Background Various experimental techniques yield peptides that are biologically active but have unfavourable pharmacological properties. The design of structurally similar organic compounds, i.e. peptide mimetics, is a challenging field in medicinal chemistry. Results SuperMimic identifies compounds that mimic parts of a protein, or positions in proteins that are suitable for inserting mimetics. The application provides libraries that contain peptidomimetic building blocks on the one hand and protein structures on the other. The search for promising peptidomimetic linkers for a given peptide is based on the superposition of the peptide with several conformers of the mimetic. New synthetic elements or proteins can be imported and used for searching. Conclusion We present a graphical user interface for finding peptide mimetics that can be inserted into a protein or for fitting small molecules into a protein. Using SuperMimic, promising locations in proteins for the insertion of mimetics can be found quickly and conveniently. PMID:16403211

  4. Template-Directed Ligation of Peptides to Oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.

    1996-01-01

    Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

  5. Conformational Restriction of Peptides Using Dithiol Bis-Alkylation.

    PubMed

    Peraro, L; Siegert, T R; Kritzer, J A

    2016-01-01

    Macrocyclic peptides are highly promising as inhibitors of protein-protein interactions. While many bond-forming reactions can be used to make cyclic peptides, most have limitations that make this chemical space challenging to access. Recently, a variety of cysteine alkylation reactions have been used in rational design and library approaches for cyclic peptide discovery and development. We and others have found that this chemistry is versatile and robust enough to produce a large variety of conformationally constrained cyclic peptides. In this chapter, we describe applications, methods, mechanistic insights, and troubleshooting for dithiol bis-alkylation reactions for the production of cyclic peptides. This method for efficient solution-phase macrocyclization is highly useful for the rapid production and screening of loop-based inhibitors of protein-protein interactions. PMID:27586339

  6. Peptide inhibitors of botulinum neurotoxin by mRNA display

    SciTech Connect

    Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H. . E-mail: yangdc@georgetown.edu

    2005-10-07

    Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs.

  7. Separation of a set of peptide sequence isomers using differential ion mobility spectrometry.

    PubMed

    Shvartsburg, Alexandre A; Creese, Andrew J; Smith, Richard D; Cooper, Helen J

    2011-09-15

    Protein identification in bottom-up proteomics requires disentangling isomers of proteolytic peptides, a major class of which are sequence inversions. Their separation using ion mobility spectrometry (IMS) has been limited to isomeric pairs. Here we demonstrate baseline separation of all seven 8-mer tryptic peptide isomers using differential IMS. Evaluation of peak capacity implies that even larger libraries should be resolved for heavier peptides with higher charge states.

  8. Separation of a Set of Peptide Sequence Isomers Using Differential Ion Mobility Spectrometry

    SciTech Connect

    Shvartsburg, Alexandre A.; Creese, Andrew J.; Smith, Richard D.; Cooper, Helen J.

    2011-08-15

    Protein identification in bottom-up proteomics requires disentangling isomers of proteolytic peptides, a major class of which are sequence inversions. Separation of sequence isomers using ion mobility spectrometry (IMS) has been reported, but limited to pairs of species. Here we demonstrate baseline separation of all seven sequences for a tryptic peptide with eight residues using differential IMS or FAIMS. Evaluations of peak capacity of the method indicate that even larger libraries should generally be separated for heavier peptides with higher charge states.

  9. Correctional Libraries, Library Standards, and Diversity.

    ERIC Educational Resources Information Center

    Shirley, Glennor L.

    2003-01-01

    Respondents to a survey of prison librarians (n=35) regarding how they cope with demands for materials and services related to diversity issues felt that adherence to Library Standards for Adult Correctional Institutions was often impeded by security concerns. Racial differences between library providers and prisoners was not considered relevant.…

  10. Art Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

    International Federation of Library Associations, The Hague (Netherlands).

    Papers on art libraries and information services for the arts, which were presented at the 1983 International Federation of Library Associations (IFLA) conference, include: (1) "'I See All': Information Technology and the Universal Availability of Images" by Philip Pacey (United Kingdom); (2) "Online Databases in the Fine Arts" by Michael Rinehart…

  11. Administrative Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

    International Federation of Library Associations, The Hague (Netherlands).

    Six papers on the theme, "Administrative Libraries in a Technological World: Past, Present, and Future--A General Stocktaking and a State of the Art Report" focus on West German telecommunications and information technology and the use of data processing and other technological aids in West German administrative libraries. These papers, which were…

  12. Israeli Special Libraries

    ERIC Educational Resources Information Center

    Foster, Barbara

    1974-01-01

    Israel is sprinkled with a noteworthy representation of special libraries which run the gamut from modest kibbutz efforts to highly technical scientific and humanities libraries. A few examples are discussed here. (Author/CH)

  13. National Library of Medicine

    MedlinePlus

    ... ideas about NLM's third century, the future of big data, and the role of libraries in supporting research ... ideas about NLM's third century, the future of big data, and the role of libraries in supporting research ...

  14. Facility Focus: Libraries.

    ERIC Educational Resources Information Center

    College Planning & Management, 2003

    2003-01-01

    Describes the designs of the Ferris State University Library for Information, Technology and Education (FLITE), and the Meyer Library and Information Technology Center at Southwest Missouri State University. Includes photographs. (EV)

  15. Reutilizing Existing Library Space.

    ERIC Educational Resources Information Center

    Davis, Marlys Cresap

    1987-01-01

    This discussion of the reutilization of existing library space reviews the decision process and considerations for implementation. Two case studies of small public libraries which reassigned space to better use are provided, including floor plans. (1 reference) (MES)

  16. Selecting Library Furniture & Equipment.

    ERIC Educational Resources Information Center

    Media & Methods, 1997

    1997-01-01

    Offers suggestions for selecting school library furniture and equipment. Describes various models of computer workstations; reading tables and chairs; and shelving. Sidebar lists names and addresses of library furniture manufactures and distributors. (AEF)

  17. FMRFamide-related peptides (FaRPs): A new family of peptides from amphibian defensive skin secretions.

    PubMed

    Wang, Lei; Smyth, Anita; Johnsen, Anders H; Zhou, Mei; Chen, Tianbao; Walker, Brian; Shaw, Chris

    2009-06-01

    Amphibian defensive skin secretions are known to contain a plethora of biologically-active peptides that are often structural and functional analogues of vertebrate neuropeptides. Here we report the structures of two invertebrate neuropeptide analogues, IPPQFMRF amide (IF-8 amide) and EGDEDEFLRF amide (EF-10 amide), from the defensive skin secretions of two different species of African hyperoliid frogs, Kassina maculata and Phylictimantis verrucosus, respectively. These represent the first canonical FMRF amide-related peptides (FaRPs) from a vertebrate source. The cDNA encoding IF-8 amide was cloned from a skin secretion library and found to contain a single copy of the peptide located at the C-terminus of a 58 amino acid residue open-reading frame. These data extend the potential targets of the defensive arsenal of amphibian tegumental secretions to parasitic/predatory invertebrates and the novel peptides described may represent the first vertebrate peptidic endectocides. PMID:19358831

  18. Brain natriutetic peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/007509.htm Brain natriuretic peptide test To use the sharing features on this page, please enable JavaScript. Brain natriuretic peptide (BNP) test is a blood test that measures ...

  19. Vasoactive intestinal peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/003508.htm Vasoactive intestinal peptide test To use the sharing features on this page, please enable JavaScript. Vasoactive intestinal peptide (VIP) is a test that measures the amount ...

  20. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested. PMID:27145593

  1. xComb: a cross-linked peptide database approach to protein-protein interaction analysis

    PubMed Central

    Panchaud, Alexandre; Singh, Pragya; Shaffer, Scott A.; Goodlett, David R.

    2010-01-01

    We developed an informatic method to identify tandem mass spectra composed of chemically cross-linked peptides from those of linear peptides and to assign sequence to each of the two unique peptide sequences. For a given set of proteins the key software tool, xComb, combs through all theoretically feasible cross-linked peptides to create a database consisting of a subset of all combinations represented as peptide FASTA files. The xComb library of select theoretical cross-linked peptides may then be used as a database that is examined by a standard proteomic search engine to match tandem mass spectral datasets to identify cross-linked peptides. The database search may be conducted against as many as 50 proteins with a number of common proteomic search engines, e.g. Phenyx, Sequest, OMSSA, Mascot and X!Tandem. By searching against a peptide library of linearized, cross-linked peptides, rather than a linearized protein library, search times are decreased and the process is decoupled from any specific search engine. A further benefit of decoupling from the search engine is that protein cross-linking studies may be conducted with readily available informatics tools for which scoring routines already exist within the proteomic community. PMID:20302351

  2. Public Libraries Going Green

    ERIC Educational Resources Information Center

    Miller, Kathryn

    2010-01-01

    Going green is now a national issue, and patrons expect their library to respond in the same way many corporations have. Libraries are going green with logos on their Web sites, programs for the public, and a host of other initiatives. This is the first book to focus strictly on the library's role in going green, helping you with: (1) Collection…

  3. Reforming Prison Libraries.

    ERIC Educational Resources Information Center

    Coyle, William J.

    1989-01-01

    Discusses the current widespread acceptance of the public library model for prison libraries, in which preferences of the inmates are the chief consideration in programing and collection development. It is argued that this model results in recreational programs and collections that fail to fulfill the prison library's role in education and…

  4. PAL: Positional Astronomy Library

    NASA Astrophysics Data System (ADS)

    Jenness, T.; Berry, D. S.

    2016-06-01

    The PAL library is a partial re-implementation of Pat Wallace's popular SLALIB library written in C using a Gnu GPL license and layered on top of the IAU's SOFA library (or the BSD-licensed ERFA) where appropriate. PAL attempts to stick to the SLA C API where possible.

  5. Creating a Classroom Library.

    ERIC Educational Resources Information Center

    Hepler, Susan; And Others

    1992-01-01

    Presents ideas for creating classroom libraries, noting how to set up a library (create a space, build and organize the collection, and set rules), where to find books at bargain prices (e.g., garage sales, libraries, book clubs, and grants), basic books to include, and information on authors and illustrators. (SM)

  6. Marketing Academic Libraries

    ERIC Educational Resources Information Center

    Mallon, Melissa, Ed.

    2013-01-01

    Ask any academic librarian if marketing their library and its services is an important task, and the answer will most likely be a resounding "yes!" Particularly in economically troubled times, librarians are increasingly called upon to promote their services and defend their library's worth. Since few academic libraries have in-house marketing…

  7. The Library Morphs

    ERIC Educational Resources Information Center

    Waters, John K.

    2008-01-01

    As campus renovation projects go, the Ohio State University's plan to turn its main library into "a library for the 21st century" is ambitious. The author describes the decade-long, $109 million transformation of the William Oxley Thompson Memorial Library. The overhaul calls for a complete replacement of all mechanical and electrical systems,…

  8. The Electronic, Eclectic Library.

    ERIC Educational Resources Information Center

    Dowlin, Kenneth E.

    1980-01-01

    Utilization of telecommunications and computer technology to increase access to information is identified as a primary goal for public libraries. To further service in the future, libraries should consider database services, online access to library resources, online community conferencing, community databases, network resource sharing,…

  9. A Truly Bookless Library

    ERIC Educational Resources Information Center

    Kolowich, Steve

    2011-01-01

    The difference between the University of Texas at San Antonio's Applied Engineering and Technology Library and other science-focused libraries is not that its on-site collection is also available electronically. It is that its on-site collection is only available electronically. The idea of libraries with no bound books has been a recurring theme…

  10. Summer Library Reading Programs

    ERIC Educational Resources Information Center

    Fiore, Carole D.

    2007-01-01

    Virtually all public libraries in the United States provide some type of summer library reading program during the traditional summer vacation period. Summer library reading programs provide opportunities for students of many ages and abilities to practice their reading skills and maintain skills that are developed during the school year. Fiore…

  11. Worthington Libraries, OH

    ERIC Educational Resources Information Center

    Berry, John N., III

    2007-01-01

    Worthington, Ohio, has deep library roots. A library has been part of its history since the planning by settlers before the city's birth in 1803. Among the treasures brought by James Kilbourne and the Scioto Company from Connecticut to the new, planned community they built was a collection of books for their new subscription library. The books…

  12. A Library That Swings

    ERIC Educational Resources Information Center

    Spragens, Thomas A.

    1971-01-01

    The Grace Doherty Library (Centre College, Danville, Kentucky) is a library that "swings," being so designed that a substantial part of its space is used alternately for classroom purposes during the day and for library reading-study space in the peak evening hours. (Author)

  13. Spanish Museum Libraries Network.

    ERIC Educational Resources Information Center

    Lopez de Prado, Rosario

    This paper describes the creation of an automated network of museum libraries in Spain. The only way in which the specialized libraries in the world today can continue to be active and to offer valid information is to automate the service they offer, and create network libraries with cooperative plans. The network can be configured with different…

  14. Place as Library?

    ERIC Educational Resources Information Center

    Davenport, Nancy

    2006-01-01

    Digital technology is redrawing the library's blueprint. Planners are thinking in new ways about how to design libraries as places for learning rather than primarily as storehouses of information. This thinking has given rise to much discussion--and to many publications--about the "library as place." In this article, the author asks why not also…

  15. The Honor System Library

    ERIC Educational Resources Information Center

    Marie, Kristen L.

    2005-01-01

    An honor system library can be created inside the library media center (LMC). Where students can access free books and magazines that require no formal checkouts. The honor library system at Washington High School, Fremont, California, has become self-sustaining. As many students, parents and teachers donate quality material. No student is ever…

  16. Technostress and Library Values.

    ERIC Educational Resources Information Center

    Gorman, Michael

    2001-01-01

    Discusses information overload and society's and libraries' responses to technology. Considers eight values that libraries should focus on and how they relate to technology in libraries: democracy, stewardship, service, intellectual freedom, privacy, rationalism, equity of access, and building harmony and balance. (LRW)

  17. Library Journal Classics.

    ERIC Educational Resources Information Center

    Crunden, Frederick; And Others

    1993-01-01

    Includes four articles originally published in the 1890s. Highlights include a discussion of freedom of speech, political opinions, and the role of the public library in social reform; the establishment of a congressional or national library; censorship in libraries; and a criticism of a librarian by his assistant. (LRW)

  18. School Libraries and Innovation

    ERIC Educational Resources Information Center

    McGrath, Kevin G.

    2015-01-01

    School library programs have measured success by improved test scores. But how do next-generation school libraries demonstrate success as they strive to be centers of innovation and creativity? These libraries offer solutions for school leaders who struggle to restructure existing systems built around traditional silos of learning (subjects and…

  19. Simple Library Bookkeeping.

    ERIC Educational Resources Information Center

    Hoffman, Herbert H.

    A simple and cheap manual double entry continuous transaction posting system with running balances is developed for bookkeeping by small libraries. A very small library may operate without any system of fiscal control but when a library's budget approaches three figures, some kind of bookkeeping must be introduced. To maintain control over his…

  20. Changing State Digital Libraries

    ERIC Educational Resources Information Center

    Pappas, Marjorie L.

    2006-01-01

    Research has shown that state virtual or digital libraries are evolving into websites that are loaded with free resources, subscription databases, and instructional tools. In this article, the author explores these evolving libraries based on the following questions: (1) How user-friendly are the state digital libraries?; (2) How do state digital…

  1. The Library Lobby

    ERIC Educational Resources Information Center

    Ralston, Anthony

    1971-01-01

    The library is examined as a university-wide resource and as a research facility. The use of the library relative to computing facilities is also considered as is the cost and value of library usage in relation to computer usage. (Author)

  2. Merchandising Your Library.

    ERIC Educational Resources Information Center

    Sivulich, Kenneth G.

    1989-01-01

    Discusses library circulation figures as a reflection of the success of library services and describes merchandising techniques that have produced a 137 percent circulation increase at Queens Borough Public Library over the past seven years. Merchandising techniques such as minibranches, displays, signage, dumps, and modified shelving are…

  3. Library Building and Design.

    ERIC Educational Resources Information Center

    Jones, David J.; Gordon, Heather; Caddy, Julie; Kahlert, Maureen; Johnson, Carolyn; Holdstock, Fiona

    1997-01-01

    More frequently, community connections are being expressed in library design briefs and reflected in the completed buildings. This collection of brief articles discusses community involvement in library design and services and describes library construction projects in Australia and Malaysia. Also, discusses community art programs, integrating…

  4. Public Library Finance.

    ERIC Educational Resources Information Center

    Mason, Marilyn Gell

    This study reviews trends in public library finance; examines recent political, economic, and technological changes; and assesses the impact of these changes on public library services. A history of the public library in America is presented, as well as an analysis of the principles of economics and public finance which reveals that current…

  5. Library Studies I Workbook.

    ERIC Educational Resources Information Center

    Frost, William J.

    Developed for use in the Library Studies I component of the Library Studies Program at Bloomsburg University (Pennsylvania), this self-paced workbook is intended to acquaint students with the Harvey A. Andruss Library and help them develop information-seeking skills. The workbook is designed to be used in conjunction with an exercise book, and…

  6. Supervision in Libraries.

    ERIC Educational Resources Information Center

    Bailey, Martha J.

    Although the literature of library administration draws extensively on that of business management, it is difficult to compare library supervision to business or industrial supervision. Library supervisors often do not have managerial training and may consider their management role as secondary. The educational level of the staff they supervise…

  7. Libraries and the Environment.

    ERIC Educational Resources Information Center

    LaRue, James; And Others

    1991-01-01

    Three articles address issues that relate to libraries and the environment. Highlights include recycling projects; buying recycled paper products and other ecology-minded purchasing ideas; energy-efficient libraries; indoor pollution problems; a list of environmental information sources; designing library buildings; and activities that libraries…

  8. Antimicrobial Peptides in 2014

    PubMed Central

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  9. PH dependent adhesive peptides

    DOEpatents

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  10. Artificial neural network study on organ-targeting peptides.

    PubMed

    Jung, Eunkyoung; Kim, Junhyoung; Choi, Seung-Hoon; Kim, Minkyoung; Rhee, Hokyoung; Shin, Jae-Min; Choi, Kihang; Kang, Sang-Kee; Lee, Nam Kyung; Choi, Yun-Jaie; Jung, Dong Hyun

    2010-01-01

    We report a new approach to studying organ targeting of peptides on the basis of peptide sequence information. The positive control data sets consist of organ-targeting peptide sequences identified by the peroral phage-display technique for four organs, and the negative control data are prepared from random sequences. The capacity of our models to make appropriate predictions is validated by statistical indicators including sensitivity, specificity, enrichment curve, and the area under the receiver operating characteristic (ROC) curve (the ROC score). VHSE descriptor produces statistically significant training models and the models with simple neural network architectures show slightly greater predictive power than those with complex ones. The training and test set statistics indicate that our models could discriminate between organ-targeting and random sequences. We anticipate that our models will be applicable to the selection of organ-targeting peptides for generating peptide drugs or peptidomimetics.

  11. Mapping protease substrates using a biotinylated phage substrate library.

    SciTech Connect

    Scholle, M. D.; Kriplani, U.; Pabon, A.; Sishtla, K.; Glucksman, M. J.; Kay, B. K.; Biosciences Division; Chicago Medical School

    2005-05-05

    We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobic is the scissile bond.

  12. Expansion of the ion library for mining SWATH-MS data through fractionation proteomics.

    PubMed

    Zi, Jin; Zhang, Shenyan; Zhou, Ruo; Zhou, Baojin; Xu, Shaohang; Hou, Guixue; Tan, Fengji; Wen, Bo; Wang, Quanhui; Lin, Liang; Liu, Siqi

    2014-08-01

    The strategy of sequential window acquisition of all theoretical fragment ion spectra (SWATH) is emerging in the field of label-free proteomics. A critical consideration for the processing of SWATH data is the quality of the ion library (or mass spectrometric reference map). As the availability of open spectral libraries that can be used to process SWATH data is limited, most users currently create their libraries in-house. Herein, we propose an approach to construct an expanded ion library using the data-dependent acquisition (DDA) data generated by fractionation proteomics. We identified three critical elements for achieving a satisfactory ion library during the iterative process of our ion library expansion, including a correction of the retention times (RTs) gained from fractionation proteomics, appropriate integrations of the fractionated proteomics into an ion library, and assessments of the impact of the expanded ion libraries to data mining in SWATH. Using a bacterial lysate as an evaluation material, we employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate the lysate proteins and constructed the expanded ion library using the fractionation proteomics data. Compared with the ion library built from the unfractionated proteomics, approximately 20% more peptides were extracted from the expanded ion library. The extracted peptides, moreover, were acceptable for further quantitative analysis.

  13. Specific phage-displayed peptides discriminate different forms of neurocysticercosis by antibody detection in the serum samples.

    PubMed

    Manhani, M N; Ribeiro, V S; Cardoso, R; Ueira-Vieira, C; Goulart, L R; Costa-Cruz, J M

    2011-06-01

    Neurocysticercosis (NC), caused by Taenia solium metacestode, infects the central nervous system and is a devastating parasitic infection. Diagnosis is based on symptoms, imaging, serology and epidemiology. Current markers present variable sensitivity and specificity, frequent cross-reactions and are not able to discriminate NC clinical forms. The aim of this study was to select mimotopes of T. solium metacestode antigens that may be used in NC immunodiagnosis, specifically to discriminate between active and inactive forms. A random peptide phage display library was screened against IgY from chickens immunized with total saline extract from T. solium metacestodes and validated against 110 serum samples, classified into active NC (18), inactive NC (22), cross-reactive parasitic diseases (40) and healthy controls (30). We have successfully selected seven peptides with significant immunoreactivity to IgG of NC patients, with sensitivity ranging from 95.5% to 100% to detect the inactive form and specificity varied from 85.7% to 94.3%. One phage-displayed peptide (Cc48) can be directly used as biomarker to distinguish inactive from active forms with an accuracy of 95.7%, and this novel mimotope may also be used as an auxiliary tool to neuroimaging tests and treatment follow-up.

  14. Peptide receptor radionuclide therapy: an overview.

    PubMed

    Dash, Ashutosh; Chakraborty, Sudipta; Pillai, Maroor Raghavan Ambikalmajan; Knapp, Furn F Russ

    2015-03-01

    Peptide receptor radionuclide therapy (PRRT) is a site-directed targeted therapeutic strategy that specifically uses radiolabeled peptides as biological targeting vectors designed to deliver cytotoxic levels of radiation dose to cancer cells, which overexpress specific receptors. Interest in PRRT has steadily grown because of the advantages of targeting cellular receptors in vivo with high sensitivity as well as specificity and treatment at the molecular level. Recent advances in molecular biology have not only stimulated advances in PRRT in a sustainable manner but have also pushed the field significantly forward to several unexplored possibilities. Recent decades have witnessed unprecedented endeavors for developing radiolabeled receptor-binding somatostatin analogs for the treatment of neuroendocrine tumors, which have played an important role in the evolution of PRRT and paved the way for the development of other receptor-targeting peptides. Several peptides targeting a variety of receptors have been identified, demonstrating their potential to catalyze breakthroughs in PRRT. In this review, the authors discuss several of these peptides and their analogs with regard to their applications and potential in radionuclide therapy. The advancement in the availability of combinatorial peptide libraries for peptide designing and screening provides the capability of regulating immunogenicity and chemical manipulability. Moreover, the availability of a wide range of bifunctional chelating agents opens up the scope of convenient radiolabeling. For these reasons, it would be possible to envision a future where the scope of PRRT can be tailored for patient-specific application. While PRRT lies at the interface between many disciplines, this technology is inextricably linked to the availability of the therapeutic radionuclides of required quality and activity levels and hence their production is also reviewed.

  15. Planning & Urban Affairs Library Manual.

    ERIC Educational Resources Information Center

    Knobbe, Mary L., Ed.; Lessel, Janice W., Ed.

    Written especially for persons without a library degree who are operating a small urban study or planning agency library on a part-time basis. Subjects covered are: (1) library function and staff function, duties and training; (2) physical layout and equipment of library; (3) establishing and maintaining the library; (4) library administration;…

  16. 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.

  17. MS FORTRAN Extended Libraries

    1986-09-01

    DISPPAK is a set of routines for use with Microsoft FORTRAN programs that allows the flexible display of information on the screen of an IBM PC in both text and graphics modes. The text mode routines allow the cursor to be placed at an arbitrary point on the screen and text to be displayed at the cursor location, making it possible to create menus and other structured displays. A routine to set the color ofmore » the characters that these routines display is also provided. A set of line drawing routines is included for use with IBM''s Color Graphics Adapter or an equivalent board (such as the Enhanced Graphics Adapter in CGA emulation mode). These routines support both pixel coordinates and a user-specified set of real number coordinates. SUBPAK is a function library which allows Microsoft FORTRAN programs to calculate random numbers, issue calls to the operating system, read individual characters from the keyboard, perform Boolean and shift operations, and communicate with the I/O ports of the IBM PC. In addition, peek and poke routines, a routine that returns the address of any variable, and routines that can access the system time and date are included.« less

  18. Comprehensive peptidomimetic libraries targeting protein-protein interactions.

    PubMed

    Whitby, Landon R; Boger, Dale L

    2012-10-16

    Transient protein-protein interactions (PPIs) are essential components in cellular signaling pathways as well as in important processes such as viral infection, replication, and immune suppression. The unknown or uncharacterized PPIs involved in such interaction networks often represent compelling therapeutic targets for drug discovery. To date, however, the main strategies for discovery of small molecule modulators of PPIs are typically limited to structurally characterized targets. Recent developments in molecular scaffolds that mimic the side chain display of peptide secondary structures have yielded effective designs, but few screening libraries of such mimetics are available to interrogate PPI targets. We initiated a program to prepare a comprehensive small molecule library designed to mimic the three major recognition motifs that mediate PPIs (α-helix, β-turn, and β-strand). Three libraries would be built around templates designed to mimic each such secondary structure and substituted with all triplet combinations of groups representing the 20 natural amino acid side chains. When combined, the three libraries would contain a member capable of mimicking the key interaction and recognition residues of most targetable PPIs. In this Account, we summarize the results of the design, synthesis, and validation of an 8000 member α-helix mimetic library and a 4200 member β-turn mimetic library. We expect that the screening of these libraries will not only provide lead structures against α-helix- or β-turn-mediated protein-protein or peptide-receptor interactions, even if the nature of the interaction is unknown, but also yield key insights into the recognition motif (α-helix or β-turn) and identify the key residues mediating the interaction. Consistent with this expectation, the screening of the libraries against p53/MDM2 and HIV-1 gp41 (α-helix mimetic library) or the opioid receptors (β-turn mimetic library) led to the discovery of library members expected

  19. Flight Software Math Library

    NASA Technical Reports Server (NTRS)

    McComas, David

    2013-01-01

    The flight software (FSW) math library is a collection of reusable math components that provides typical math utilities required by spacecraft flight software. These utilities are intended to increase flight software quality reusability and maintainability by providing a set of consistent, well-documented, and tested math utilities. This library only has dependencies on ANSI C, so it is easily ported. Prior to this library, each mission typically created its own math utilities using ideas/code from previous missions. Part of the reason for this is that math libraries can be written with different strategies in areas like error handling, parameters orders, naming conventions, etc. Changing the utilities for each mission introduces risks and costs. The obvious risks and costs are that the utilities must be coded and revalidated. The hidden risks and costs arise in miscommunication between engineers. These utilities must be understood by both the flight software engineers and other subsystem engineers (primarily guidance navigation and control). The FSW math library is part of a larger goal to produce a library of reusable Guidance Navigation and Control (GN&C) FSW components. A GN&C FSW library cannot be created unless a standardized math basis is created. This library solves the standardization problem by defining a common feature set and establishing policies for the library s design. This allows the libraries to be maintained with the same strategy used in its initial development, which supports a library of reusable GN&C FSW components. The FSW math library is written for an embedded software environment in C. This places restrictions on the language features that can be used by the library. Another advantage of the FSW math library is that it can be used in the FSW as well as other environments like the GN&C analyst s simulators. This helps communication between the teams because they can use the same utilities with the same feature set and syntax.

  20. Assessing Library Automation and Virtual Library Development in Four Academic Libraries in Oyo, Oyo State, Nigeria

    ERIC Educational Resources Information Center

    Gbadamosi, Belau Olatunde

    2011-01-01

    The paper examines the level of library automation and virtual library development in four academic libraries. A validated questionnaire was used to capture the responses from academic librarians of the libraries under study. The paper discovers that none of the four academic libraries is fully automated. The libraries make use of librarians with…

  1. Confident site localization using a simulated phosphopeptide spectral library.

    PubMed

    Suni, Veronika; Imanishi, Susumu Y; Maiolica, Alessio; Aebersold, Ruedi; Corthals, Garry L

    2015-05-01

    We have investigated if phosphopeptide identification and simultaneous site localization can be achieved by spectral library searching. This allows taking advantage of comparison of specific spectral features, which would lead to improved discrimination of differential localizations. For building a library, we propose a spectral simulation strategy where all possible single phosphorylations can be simply and accurately (re)constructed on enzymatically dephosphorylated peptides, by predicting the diagnostic fragmentation events produced in beam-type CID. To demonstrate the performance of our approach, enriched HeLa phosphopeptides were dephosphorylated with alkaline phosphatase and analyzed with higher energy collisional dissociation (HCD), which were then used for creating a spectral library of simulated phosphopeptides. Spectral library searching using SpectraST was performed on data sets of synthetic phosphopeptides and the HeLa phosphopeptides, and subsequently compared to Mascot and Sequest database searching followed by phosphoRS and Ascore afforded localization, respectively. Our approach successfully led to accurate localization, and it outperformed other methods, when phosphopeptides were covered by the library. These results suggest that the searching with simulated spectral libraries serves as a crucial approach for both supplementing and validating the phosphorylation sites obtained by database searching and localization tools. For future development, simulation of multiply phosphorylated peptides remains to be implemented.

  2. 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.

  3. In vitro affinity screening of protein and peptide binders by megavalent bead surface display.

    PubMed

    Diamante, Letizia; Gatti-Lafranconi, Pietro; Schaerli, Yolanda; Hollfelder, Florian

    2013-10-01

    The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead K(d) measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).

  4. Case studies on the development of ScaLAPACK and the NAG numerical PVM library

    SciTech Connect

    Dongarra, J.J. |; Hammarling, S. |; Petitet, A.

    1996-12-31

    In this paper we look at the development of ScaLAPACK, a software library for dense and banded numerical linear algebra, and the NAG Numerical PVM Library, which includes software for dense and sparse linear algebra, quadrature, optimization and random number generation. Both libraries are aimed at distributed memory machines, including networks of workstations. The paper concentrates on the underlying design and the testing of the libraries.

  5. HER2 Targeting Peptides Screening and Applications in Tumor Imaging and Drug Delivery

    PubMed Central

    Geng, Lingling; Wang, Zihua; Jia, Xiangqian; Han, Qiuju; Xiang, Zhichu; Li, Dan; Yang, Xiaoliang; Zhang, Di; Bu, Xiangli; Wang, Weizhi; Hu, Zhiyuan; Fang, Qiaojun

    2016-01-01

    Herein, computational-aided one-bead-one-compound (OBOC) peptide library design combined with in situ single-bead sequencing microarray methods were successfully applied in screening peptides targeting at human epidermal growth factor receptor-2 (HER2), a biomarker of human breast cancer. As a result, 72 novel peptides clustered into three sequence motifs which are PYL***NP, YYL***NP and PPL***NP were acquired. Particularly one of the peptides, P51, has nanomolar affinity and high specificity for HER2 in ex vivo and in vivo tests. Moreover, doxorubicin (DOX)-loaded liposome nanoparticles were modified with peptide P51 or P25 and demonstrated to improve the targeted delivery against HER2 positive cells. Our study provides an efficient peptide screening method with a combination of techniques and the novel screened peptides with a clear binding site on HER2 can be used as probes for tumor imaging and targeted drug delivery. PMID:27279916

  6. Engineering a Transmembrane Nanopore Ion Channel from a Membrane Breaker Peptide.

    PubMed

    Lella, Muralikrishna; Mahalakshmi, Radhakrishnan

    2016-07-01

    Re-engineering nature's molecules is an ideal strategy to obtain explicit functionality such as synthetic molecular machines, yet novel strategies for producing engineered molecular channels are few. Here we report a peptide engineering strategy through sequence reversal, which we applied on the first transmembrane peptide of the mycobacteriophage membranoporin protein holin. We have successfully redesigned the membrane rupture property of this peptide to form specific nanopore ion channels. We report the structural characterization and electrophysiology measurements of a library of 28-residue engineered membrane peptides, with remarkable ion channel behavior. We further identify that key residues at the peptide terminus, the central proline, charge distribution, and hydropathy index of the peptide together contribute to the channel properties that we measure. Our sequence reversal strategy for peptide engineering to successfully obtain nanopore channels can pave the way for better biobased design of controlled nanopores, using only natural amino acids. PMID:27257735

  7. HER2 Targeting Peptides Screening and Applications in Tumor Imaging and Drug Delivery.

    PubMed

    Geng, Lingling; Wang, Zihua; Jia, Xiangqian; Han, Qiuju; Xiang, Zhichu; Li, Dan; Yang, Xiaoliang; Zhang, Di; Bu, Xiangli; Wang, Weizhi; Hu, Zhiyuan; Fang, Qiaojun

    2016-01-01

    Herein, computational-aided one-bead-one-compound (OBOC) peptide library design combined with in situ single-bead sequencing microarray methods were successfully applied in screening peptides targeting at human epidermal growth factor receptor-2 (HER2), a biomarker of human breast cancer. As a result, 72 novel peptides clustered into three sequence motifs which are PYL***NP, YYL***NP and PPL***NP were acquired. Particularly one of the peptides, P51, has nanomolar affinity and high specificity for HER2 in ex vivo and in vivo tests. Moreover, doxorubicin (DOX)-loaded liposome nanoparticles were modified with peptide P51 or P25 and demonstrated to improve the targeted delivery against HER2 positive cells. Our study provides an efficient peptide screening method with a combination of techniques and the novel screened peptides with a clear binding site on HER2 can be used as probes for tumor imaging and targeted drug delivery. PMID:27279916

  8. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions.

  9. Plant peptide hormone signalling.

    PubMed

    Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

    2015-01-01

    The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions. PMID:26374891

  10. Combinatorial approaches: A new tool to search for highly structured β-hairpin peptides

    PubMed Central

    Pastor, Maria Teresa; López de la Paz, Manuela; Lacroix, Emmanuel; Serrano, Luis; Pérez-Payá, Enrique

    2002-01-01

    Here we present a combinatorial approach to evolve a stable β-hairpin fold in a linear peptide. Starting with a de novo-designed linear peptide that shows a β-hairpin structure population of around 30%, we selected four positions to build up a combinatorial library of 204 sequences. Deconvolution of the library using circular dichroism reduced such a sequence complexity to 36 defined sequences. Circular dichroism and NMR of these peptides resulted in the identification of two linear 14-aa-long peptides that in plain buffered solutions showed a percentage of β-hairpin structure higher than 70%. Our results show how combinatorial approaches can be used to obtain highly structured peptide sequences that could be used as templates in which functionality can be introduced. PMID:11782528

  11. Engineering and analysis of peptide-recognition domain specificities by phage display and deep sequencing.

    PubMed

    McLaughlin, Megan E; Sidhu, Sachdev S

    2013-01-01

    Protein interaction networks depend in part on the specific recognition of unstructured peptides by folded domains. Understanding how members of a domain family use a similar fold to recognize different peptide sequences selectively is a fundamental question. One way to advance our understanding of peptide recognition is to apply an existing model of peptide recognition for a particular domain toward engineering synthetic domain variants with desired properties. Successes, failures, and unintended outcomes can help refine the model and can illuminate more general principles of peptide recognition. Using the PDZ domain fold as an example, we describe methods for (1) structure-based combinatorial library design and directed evolution of domain variants and (2) specificity profiling of large repertoires of synthetic variants using multiplexed deep sequencing. Peptide-binding preferences for hundreds of variants can be decoded in parallel, enabling comparisons between different library designs and selection pressures. The tremendous depth of coverage of the binding peptide profiles also permits robust computational analysis. This approach to studying peptide recognition can be applied to other domains and to a variety of structural and functional models by tailoring the combinatorial library design and selection pressures accordingly.

  12. I.D. Weeks Library: Guide to Library Resources.

    ERIC Educational Resources Information Center

    South Dakota Univ., Vermillion. I.D. Weeks Library.

    This document is a guide to library resources in the I. D. Weeks Library at the University of South Dakota, Vermillion. The guide was designed to accompany an academic library instruction course, "Use of Library Resources, A & S 111." The purpose of the course is to familiarize students with the facilities of the I. D. Weeks Library; to acquaint…

  13. Libraries are for People! But Are the People for Libraries?

    ERIC Educational Resources Information Center

    Ohio State Library, Columbus.

    The 1974 Ohio Governor's Conference on Library and Information Services was the first in a series of meetings mandated by the Long Range Program for the Improvement of Library Services developed by the State Library Board to give citizens a voice in the future of their libraries. Information was gathered on the kinds of libraries--school,…

  14. Human 20S proteasome activity towards fluorogenic peptides of various chain lengths.

    PubMed

    Rut, Wioletta; Drag, Marcin

    2016-09-01

    The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal. PMID:27176742

  15. Human 20S proteasome activity towards fluorogenic peptides of various chain lengths.

    PubMed

    Rut, Wioletta; Drag, Marcin

    2016-09-01

    The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal.

  16. Integrated circuit cell library

    NASA Technical Reports Server (NTRS)

    Whitaker, Sterling R. (Inventor); Miles, Lowell H. (Inventor)

    2005-01-01

    According to the invention, an ASIC cell library for use in creation of custom integrated circuits is disclosed. The ASIC cell library includes some first cells and some second cells. Each of the second cells includes two or more kernel cells. The ASIC cell library is at least 5% comprised of second cells. In various embodiments, the ASIC cell library could be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more comprised of second cells.

  17. Liquid-phase combinatorial library synthesis: recent advances and future perspectives.

    PubMed

    Barot, Kuldipsinh P; Nikolova, Stoyanka; Ivanov, Illiyan; Ghate, Manjunath D

    2014-01-01

    Liquid-phase combinatorial library synthesis is commonly developed into the viable alternatives or adjunct across the broad spectrum of polymer-supported organic chemistry. It includes the use of soluble polymer supports in the combinatorial synthesis of peptides and small-molecular library compounds which act as catalyst and reagent supports. It also includes high throughput biological screening with generation and evaluation of chemical leads for drug discovery development. In this review, liquid-phase combinatorial library synthesis is shown as the most efficient method of choice for the synthesis of most of the combinatorial library compounds with specific approaches from different groups that state potentials of solution-phase combinatorial synthesis.

  18. Antimicrobial Peptides in Reptiles

    PubMed Central

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  19. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    SciTech Connect

    Evans, D.M.; Williams, K.P.; McGuinness, B.

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  20. The Jordanian Library Scene, 1973

    ERIC Educational Resources Information Center

    International Library Review, 1974

    1974-01-01

    This discussion is made up of several individual papers by different authors: Introduction; Libraries in Jordan; The Royal Scientific Society and its Library; The Royal Scientific Society Course in Librarianship; Jordan Library Association Course in Librarianship; Secondary School Libraries in Jordan; and Notes on West Bank Libraries, 1972. (JB)

  1. Public Relations in Special Libraries.

    ERIC Educational Resources Information Center

    Rutkowski, Hollace Ann; And Others

    1991-01-01

    This theme issue includes 11 articles on public relations (PR) in special libraries. Highlights include PR at the Special Libraries Association (SLA); sources for marketing research for libraries; developing a library image; sample PR releases; brand strategies for libraries; case studies; publicizing a consortium; and a bibliography of pertinent…

  2. Standards for Utah Public Libraries.

    ERIC Educational Resources Information Center

    Utah State Library Div., Salt Lake City. Dept. of Community and Economic Development.

    This document presents standards for Utah public libraries. The benefits of standards for public libraries and for the state library are listed, and responsibilities, benefits, and characteristics of three types of public libraries (i.e., volunteer, transitional, and certified public libraries) are summarized. Specific standards in the following…

  3. Library Latchkey Children. ERIC Digest.

    ERIC Educational Resources Information Center

    Dowd, Frances Smardo

    This digest discusses ways in which public libraries deal with latchkey children who spend their after-school hours at public libraries while their parents are at work. Research conducted in 1990 of 110 public libraries revealed that almost all libraries encountered unattended children after school hours, and that most libraries surveyed were not…

  4. Ohio: Library and Information Networks.

    ERIC Educational Resources Information Center

    Byerly, Greg

    1996-01-01

    Describes the development, current status, and future goals and plans of library and information networks in Ohio. Highlights include OCLC; OhioLINK, incorporating state-assisted universities, two-year colleges, the state library, and private university libraries; a school library network; a public library information network; a telecommunications…

  5. Resources of North Carolina Libraries.

    ERIC Educational Resources Information Center

    Downs, Robert B., Ed.

    The conclusion of the survey is that North Carolina libraries do not have sufficient resources, physical facilities or staff to provide adequate library service for the state. The survey covers the present and potential roles of the State Library, the State Department of Archives and History, public libraries, university libraries, senior and…

  6. FCLib: The Feature Characterization Library.

    SciTech Connect

    Gentile, Ann C.; Doyle, Wendy S. K.; Kegelmeyer, W. Philip,; Ulmer, Craig D.

    2008-11-01

    The Feature Characterization Library (FCLib) is a software library that simplifies the process of interrogating, analyzing, and understanding complex data sets generated by finite element applications. This document provides an overview of the library, a description of both the design philosophy and implementation of the library, and examples of how the library can be utilized to extract understanding from raw datasets.

  7. Optical Disc Applications in Libraries.

    ERIC Educational Resources Information Center

    Andre, Pamela Q. J.

    1989-01-01

    Discusses a variety of library applications of optical disc storage technology, including CD-ROM, digital videodisc, and WORM. Research and development projects at the Library of Congress, National Library of Medicine, and National Agricultural Library are described, products offered by library networks are reviewed, and activities in academic and…

  8. Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo Mediated by Genetically Encoded Amino-Thiol Unnatural Amino Acids

    PubMed Central

    Frost, John R.; Jacob, Nicholas T.; Papa, Louis J.; Owens, Andrew E.

    2015-01-01

    A versatile method for orchestrating the formation of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side-chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner in vitro, or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low micromolar streptavidin binder (KD = 1.1 µM) from a library of cyclic peptides produced in E. coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles. PMID:25933125

  9. Molecular detection via hybrid peptide-semiconductor photonic devices

    NASA Astrophysics Data System (ADS)

    Estephan, E.; Saab, M.-b.; Martin, M.; Cloitre, T.; Larroque, C.; Cuisinier, F. J. G.; Malvezzi, A. M.; Gergely, C.

    2011-03-01

    The aim of this work was to investigate the possibilities to support device functionality that includes strongly confined and localized light emission and detection processes within nano/micro-structured semiconductors for biosensing applications. The interface between biological molecules and semiconductor surfaces, yet still under-explored is a key issue for improving biomolecular recognition in devices. We report on the use of adhesion peptides, elaborated via combinatorial phage-display libraries for controlled placement of biomolecules, leading to user-tailored hybrid photonic systems for molecular detection. An M13 bacteriophage library has been used to screen 1010 different peptides against various semiconductors to finally isolate specific peptides presenting a high binding capacity for the target surfaces. When used to functionalize porous silicon microcavities (PSiM) and GaAs/AlGaAs photonic crystals, we observe the formation of extremely thin (<1nm) peptide layers, hereby preserving the nanostructuration of the crystals. This is important to assure the photonic response of these tiny structures when they are functionalized by a biotinylated peptide layer and then used to capture streptavidin. Molecular detection was monitored via both linear and nonlinear optical measurements. Our linear reflectance spectra demonstrate an enhanced detection resolution via PSiM devices, when functionalized with the Si-specific peptide. Molecular capture at even lower concentrations (femtomols) is possible via the second harmonic generation of GaAs/AlGaAs photonic crystals when functionalized with GaAs-specific peptides. Our work demonstrates the outstanding value of adhesion peptides as interface linkers between semiconductors and biological molecules. They assure an enhanced molecular detection via both linear and nonlinear answers of photonic crystals.

  10. 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.

  11. Conversion of scFv peptide-binding specificity for crystal chaperone development

    SciTech Connect

    Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A.

    2012-02-07

    In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 {angstrom} resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a {approx}52 {angstrom} channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries.

  12. Surface expression, peptide repertoire, and thermostability of chicken class I molecules correlate with peptide transporter specificity

    PubMed Central

    Tregaskes, Clive A.; Harrison, Michael; Sowa, Anna K.; van Hateren, Andy; Hunt, Lawrence G.; Vainio, Olli; Kaufman, Jim

    2016-01-01

    The chicken major histocompatibility complex (MHC) has strong genetic associations with resistance and susceptibility to certain infectious pathogens. The cell surface expression level of MHC class I molecules varies as much as 10-fold between chicken haplotypes and is inversely correlated with diversity of peptide repertoire and with resistance to Marek’s disease caused by an oncogenic herpesvirus. Here we show that the average thermostability of class I molecules isolated from cells also varies, being higher for high-expressing MHC haplotypes. However, we find roughly the same amount of class I protein synthesized by high- and low-expressing MHC haplotypes, with movement to the cell surface responsible for the difference in expression. Previous data show that chicken TAP genes have high allelic polymorphism, with peptide translocation specific for each MHC haplotype. Here we use assembly assays with peptide libraries to show that high-expressing B15 class I molecules can bind a much wider variety of peptides than are found on the cell surface, with the B15 TAPs restricting the peptides available. In contrast, the translocation specificity of TAPs from the low-expressing B21 haplotype is even more permissive than the promiscuous binding shown by the dominantly expressed class I molecule. B15/B21 heterozygote cells show much greater expression of B15 class I molecules than B15/B15 homozygote cells, presumably as a result of receiving additional peptides from the B21 TAPs. Thus, chicken MHC haplotypes vary in several correlated attributes, with the most obvious candidate linking all these properties being molecular interactions within the peptide-loading complex (PLC). PMID:26699458

  13. Facile Analysis and Sequencing of Linear and Branched Peptide Boronic Acids by MALDI Mass Spectrometry

    PubMed Central

    Crumpton, Jason; Zhang, Wenyu; Santos, Webster

    2011-01-01

    Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries. PMID:21449540

  14. Multiplex localization of sequential peptide epitopes by use of a planar microbead chip.

    PubMed

    Schmidt, Carsten; Rödiger, Stefan; Gruner, Melanie; Moncsek, Anja; Stohwasser, Ralf; Hanack, Katja; Schierack, Peter; Schröder, Christian

    2016-02-18

    Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28γ (proteasome activator 28γ) polyclonal serum, for a murine monoclonal antibody against PA28γ, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks.

  15. 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

  16. Revolution and the Library.

    ERIC Educational Resources Information Center

    Gorniak-Kocikowska, Krystyna

    2001-01-01

    Considers the impact that the computer revolution has had on college and university libraries. Discusses the historical background of changes that resulted from the invention of the printing press; the development of national languages; knowledge acquisition; the historical role of religion; content control of libraries; and changes in students.…

  17. Corporations and Library Fundraising.

    ERIC Educational Resources Information Center

    DiMattia, Susan S.

    1984-01-01

    Examination of corporate donations of cash, products, service, and expertise to libraries highlights industry contributions in 1980; why corporations give; examples of corporate donations to various libraries (Brooklyn Public, New York Public, Altoona Area Public, Boston Public); planning fund-raising compaigns; and seven strategic planning…

  18. Guidelines for National Libraries.

    ERIC Educational Resources Information Center

    Sylvestre, Guy

    This report is designed to provide practical assistance to government officials and librarians responsible for the planning, creation, and development of national library services. Based on a number of authoritative studies and a broad consensus among experts, including directors of national libraries, these guidelines give special attention to…

  19. Health Science Libraries Today.

    ERIC Educational Resources Information Center

    Kenney, Brigitte L.

    This study explores means by which biomedical information might be distributed over a network to physicians and other personnel in the health sciences. Health Science libraries of all types are surveyed in terms of location, facilities, collection, staff, budget, and services. The library's user group is presented, and cooperative agreements among…

  20. Prison Libraries: Bibliography.

    ERIC Educational Resources Information Center

    Gillespie, David M.

    An Alphabetically arranged bibliography, listing 485 entries representing 518 citations taken from "Poole's Index to Periodical Literature" 1802-1906, "Cannon's Bibliography of Library Economy," and "Library Literature" 1921 to mid-1970. Other sources used include: The Index to the Journal of Correctional Education; ten bibliographies taken from…

  1. "Greening" up the Library

    ERIC Educational Resources Information Center

    Woodland, Jean

    2010-01-01

    This paper looks at the library's ability to spread environmental conservation efforts throughout the community. The paper suggests various ways library staff can explore environmentally friendly procedures within the context of the media center as well as ways to promote community interest and support. It identifies areas for improvement, ideas…

  2. Small Public Library Management

    ERIC Educational Resources Information Center

    Pearlmutter, Jane; Nelson, Paul

    2012-01-01

    Anyone at the helm of a small public library knows that every little detail counts. But juggling the responsibilities that are part and parcel of the job is far from easy. Finally, here's a handbook that includes everything administrators need to keep a handle on library operations, freeing them up to streamline and improve how the organization…

  3. Attribution of Library Costs

    ERIC Educational Resources Information Center

    Drake, Miriam A.

    1977-01-01

    Universities conduct a variety of cost-allocation studies that require the collection and analysis of the library cost-data. Cost accounting methods are used in most studies; however, costs are attributed to library user groups in a variety of ways. Cost accounting studies are reviewed and allocation methods are discussed. (Author)

  4. Guidelines on Library Twinning.

    ERIC Educational Resources Information Center

    Doyle, Robert P., Comp.; Scarry, Patricia, Comp.

    This document serves as an overview of the many forms of library twinning, or formal exchanges of materials and/or staff between two institutions. Twinning can be reciprocal, but often involves a one-way arrangement whereby a library in a developing country receives a visit from a librarian of a developed country. Either way, both institutions can…

  5. Library Technology International

    ERIC Educational Resources Information Center

    Breeding, Marshall

    2008-01-01

    The author has been extremely fortunate over the last few years to have had the opportunity to travel to many different parts of the world and speak and work with librarians in many countries and to have the chance to see first-hand some incredible libraries that demonstrate creative approaches to library services, innovative uses of technology,…

  6. Library Laws of Texas.

    ERIC Educational Resources Information Center

    Seidenberg, Ed, Ed.

    Compiled to provide a central reference point for all legislative information pertaining to libraries in the state of Texas, this publication includes all pertinent legislation as amended through the 66th Legislature, Regular Session, 1979. It contains articles dealing specifically with archives, buildings and property, city libraries, non-profit…

  7. Library Automation in Australia.

    ERIC Educational Resources Information Center

    Blank, Karen L.

    1984-01-01

    Discussion of Australia's move toward library automation highlights development of a national bibliographic network, local and regional cooperation, integrated library systems, telecommunications, and online systems, as well as microcomputer usage, ergonomics, copyright issues, and national information policy. Information technology plans of the…

  8. Hospital Library Administration.

    ERIC Educational Resources Information Center

    Cramer, Anne

    The objectives of a hospital are to improve patient care, while the objectives of a hospital library are to improve services to the staff which will support their efforts. This handbook dealing with hospital administration is designed to aid the librarian in either implementing a hospital library, or improving services in an existing medical…

  9. Library Consortia in Hungary

    ERIC Educational Resources Information Center

    Csajbok, Edit; Szluka, Peter; Vasas, Livia

    2012-01-01

    During the last two decades many Hungarian libraries have developed considerably, beyond what was considered possible prior to 1989 and the beginning of events signaling the end of Communism in the country. Some of the modernization of library services has been realized through participation in cooperative agreements. Many smaller and larger…

  10. Dumping the "Library."

    ERIC Educational Resources Information Center

    Crowley, Bill

    1998-01-01

    Discusses an alternative to abandoning the word "library" for "information" in graduate education. Recommends patient, consistent effort by library- and information-science educators to convince academic librarians that if they accept the standards of the teaching/research faculty, including the need to earn a Ph.D., it will raise the prestige of…

  11. Library Classification 2020

    ERIC Educational Resources Information Center

    Harris, Christopher

    2013-01-01

    In this article the author explores how a new library classification system might be designed using some aspects of the Dewey Decimal Classification (DDC) and ideas from other systems to create something that works for school libraries in the year 2020. By examining what works well with the Dewey Decimal System, what features should be carried…

  12. Library Journal Classics.

    ERIC Educational Resources Information Center

    Colwell, Ernest Cadman; And Others

    1993-01-01

    Includes reprints of four articles originally published in "Library Journal" in the 1940s and 1960s: "The Role of the Professional School in the University" (Ernest Cadman Colwell); "Library Schools Reshaping Courses" (Lewis F. Stieg); "The New Training Pattern Looks Good" (Harold Lancour); and "The Character and Responsibility of a Graduate…

  13. Planning for Library Excellence.

    ERIC Educational Resources Information Center

    Virginia State Library and Archives, Richmond.

    Intended to be used by librarians, this guide will also be useful to boards of trustees, governing officials, members of funding agencies, and community support groups involved in planning on a local level and within the context of regional and state library service. It provides information to help libraries plan and evaluate their services and…

  14. The Library as Ecosystem

    ERIC Educational Resources Information Center

    Walter, Scott

    2008-01-01

    Ecology is the study of interactions between organisms and their environment, and the academic library could be considered to be an ecosystem, i.e., a "biological organization" in which multiple species must interact, both with one another and with their environment. The metaphor of the library as ecosystem is flexible enough to be applied not…

  15. Privacy and Library Records

    ERIC Educational Resources Information Center

    Bowers, Stacey L.

    2006-01-01

    This paper summarizes the history of privacy as it relates to library records. It commences with a discussion of how the concept of privacy first originated through case law and follows the concept of privacy as it has affected library records through current day and the "USA PATRIOT Act."

  16. XML in Libraries.

    ERIC Educational Resources Information Center

    Tennant, Roy, Ed.

    This book presents examples of how libraries are using XML (eXtensible Markup Language) to solve problems, expand services, and improve systems. Part I contains papers on using XML in library catalog records: "Updating MARC Records with XMLMARC" (Kevin S. Clarke, Stanford University) and "Searching and Retrieving XML Records via the Web" (Theo van…

  17. Math in the Library?

    ERIC Educational Resources Information Center

    Henry, Robin

    2004-01-01

    Math and information searching skills might be effectively combined for a fun and informative unit in the library media center like the Number Hunt unit which gets students using math and library search skills to find answers to some tough numerical questions. Some of the objectives of Number Hunt Lesson plan are discussed giving a detailed…

  18. FRBR and School Libraries

    ERIC Educational Resources Information Center

    Hart, Amy

    2006-01-01

    This article discusses the Functional Requirements for Bibliographic Records (FRBR), a plan that is quickly gaining the support of library organizations around the globe. The FRBR Study Group was appointed by International Federation of Library Associations (IFLA) and issued its final report in 1998. The report redefines the basic unit of…

  19. Guide to Library Skills.

    ERIC Educational Resources Information Center

    McIntyre, C. Burnelle

    Directed toward students in freshman English courses, this guide to the basic skills in using the library is composed of three broad units: (1) orientation to the library, including computer-output microfilm, card catalogs, and location of materials; (2) reference books, including use of the dictionary, encyclopedias, and the vertical file; and…

  20. Centennial State Libraries, 1998.

    ERIC Educational Resources Information Center

    Parent, Kathleen D., Ed.

    1998-01-01

    This document consists of 12 consecutive issues of the monthly "Centennial State Libraries" newsletter, of the Colorado Department of Education, State Library and Adult Education Office. The issues cover the year 1998. Each issue of the newsletter--except the August issue which is an Annual Report--includes some or all of the following sections:…

  1. Disaster Planning in Libraries

    ERIC Educational Resources Information Center

    Wong, Yi Ling; Green, Ravonne

    2006-01-01

    Disaster preparedness is an important issue in library management today. This article presents a general overview of the theoretical aspects of disaster planning in libraries. The stages of disaster planning are a circular process of planning, prevention, response, recovery, preparedness, and training.

  2. Technology for Libraries.

    ERIC Educational Resources Information Center

    Phenix, Katharine; And Others

    1990-01-01

    Five articles discuss information technology in libraries: (1) "Software for Libraries" (Katharine Phenix); (2) "Online Update: European Online Services" (Martin Kesselman); (3) "Connect Time: Online Pricing Breakthroughs" (Barbara Quint); (4) "Microcomputing: Micro Biology Computer Viruses" (James LaRue); and (5) "Using Technology: Spreadsheet…

  3. LSCA Builds Michigan Libraries.

    ERIC Educational Resources Information Center

    Michigan Library, Lansing.

    This report highlights 19 of the 48 recently completed public library construction projects in Michigan which received partial funding from the 1983 Emergency Jobs Act. The grants were administered under Title II of the Library Services and Construction Act (LSCA). Introductory material includes listings of the State of Michigan Legislative…

  4. Library Automation Style Guide.

    ERIC Educational Resources Information Center

    Gaylord Bros., Liverpool, NY.

    This library automation style guide lists specific terms and names often used in the library automation industry. The terms and/or acronyms are listed alphabetically and each is followed by a brief definition. The guide refers to the "Chicago Manual of Style" for general rules, and a notes section is included for the convenience of individual…

  5. Libraries: Drawn to Knowledge.

    ERIC Educational Resources Information Center

    Havens, Kevin

    2003-01-01

    Discusses how to make a college library the center of campus activity, explaining how to find the proper balance of technology and tradition (e.g., harnessing new media to teach critical thinking skills, having library staff assume primary responsibility for providing information literacy training, training students to use print as well as…

  6. Homelessness in Public Libraries

    ERIC Educational Resources Information Center

    Wong, Yi Ling

    2009-01-01

    This paper takes a theoretical and practical approach in defining the "problem" of homelessness in libraries. The author examines three fundamental problems on homelessness. The three fundamental questions are: (a) Who are the homeless? (b) Why are they homeless? (c) What are their information needs in libraries? These questions are important in…

  7. Global Warming's Library Challenge

    ERIC Educational Resources Information Center

    Meyer, Jennifer

    2008-01-01

    Like every institution that uses energy, consumes resources, and engages in construction or renovation, libraries have an impact on the environment and on the critical problem of climate change. Taking action to protect library collections is not only an idealistic professional goal but also a very practical one. Disaster preparation measures and…

  8. Computer Based Library Orientation.

    ERIC Educational Resources Information Center

    Machalow, Robert

    This document presents computer-based lessons used to teach basic library skills to college students at York College of the City University of New York. The information for library orientation has been entered on a disk which must be used in conjunction with a word processing program, the Applewriter IIe, and an Apple IIe microcomputer. The…

  9. University Libraries in Transition.

    ERIC Educational Resources Information Center

    Hyatt, James A.

    1986-01-01

    College and university libraries are experiencing change in the ways they provide services and in their responses to rising costs and reduced financial support. These conditions result from three major phenomena: the information explosion, the technology revolution, and escalating library costs. (MLW)

  10. Libraries and Adult Learners.

    ERIC Educational Resources Information Center

    Josey, E. J., Ed.

    1982-01-01

    Of the 13 essays presented in this special issue on libraries and adult education, 8 focus on programs and services from the public library for adult learners. These essays provide information on: (1) an Education Information Centers Program (EIC) designed to complement employment skills training provided under the Comprehensive Employment and…

  11. The Animated Library

    ERIC Educational Resources Information Center

    Brewer, Jim; Dyal, Donald H.; Sweet, Robert

    2009-01-01

    Libraries have always been poised at the crossroads of access tools and content. Librarians, their personnel, and supporters have worked for generations to create tools to store and utilize content for the benefit of patrons. Libraries house materials and the tools to unlock them; their staffers teach patrons to use the materials and associated…

  12. Libraries at the Ready

    ERIC Educational Resources Information Center

    Celano, Donna C.; Neuman, Susan B.

    2016-01-01

    Because English language learners enter kindergarten at a distinct disadvantage, Celano and Neuman examine the role public libraries can play in rallying around these young children to better prepare them for school. The authors document a new program called Every Child Ready to Read, which recently launched in 4,000 public libraries across the…

  13. Improving Library Research Skills.

    ERIC Educational Resources Information Center

    Holley, Catherine W.

    This report describes a program to advance library research skills in two sixth grade science classes. The problem was assessed through a survey, questionnaire, and worksheet, and by direct observation. Analysis of probable cause data revealed that students displayed a lack of research skills related to library research. Some of the causes were…

  14. Maintaining Precious Library Resources.

    ERIC Educational Resources Information Center

    Wiens, Janet

    2002-01-01

    Using the examples of libraries at Southeast Missouri State University and the University of North Texas, discusses the big-picture approach and extensive communication among all users necessary for library maintenance efforts. Addresses establishing a master plan, prioritizing projects, having the right staff, and communicating. (EV)

  15. Facility Focus: Libraries.

    ERIC Educational Resources Information Center

    College Planning & Management, 1997

    1997-01-01

    Describes four operating libraries located at Union Theological Seminary in Virginia, Emory University in Georgia, Kean College in New Jersey, and Community College of Philadelphia. Their size, cost, date completed, architect, and various features are provided. Despite technological advances, the college library is a feasible part of higher…

  16. An Online Library Catalogue.

    ERIC Educational Resources Information Center

    Alloro, Giovanna; Ugolini, Donatella

    1992-01-01

    Describes the implementation of an online catalog in the library of the National Institute for Cancer Research and the Clinical and Experimental Oncology Institute of the University of Genoa. Topics addressed include automation of various library functions, software features, database management, training, and user response. (10 references) (MES)

  17. Library Resources Workbook, Revised.

    ERIC Educational Resources Information Center

    Phillips, Dennis

    Intended to provide a basic overview of the facilities and resources of the library on the Allentown Campus of Pennsylvania State University, this student workbook is designed to familiarize students with what is available to them in the library; to introduce them to skills likely to be used in varying degrees throughout life, regardless of what…

  18. Budgeting for Libraries.

    ERIC Educational Resources Information Center

    Randall, G. E.

    Empirically and rationally derived bases for determining the costs for industrial libraries are suggested. Taken into consideration is the fact that recent accounting procedures and the advent of new technologies have introduced costs into the library budget so that literature and personnel costs may now account for only 75-80 percent of the…

  19. Libraries for Small Museums.

    ERIC Educational Resources Information Center

    Anderson, Linda M.

    Presented are the very basic requirements for establishing a small special library operating under a limited budget. Physical plant organization, cataloging, book processing, circulation procedures, book selection and ordering and instructions for typists are covered. Although the practices discussed were established for a museum library, what is…

  20. Libraries & Reading: Indispensable Partners.

    ERIC Educational Resources Information Center

    Middle Grades Reading Network, Evansville, IN.

    Attention to school libraries must be at the heart of any comprehensive plan for improving youth literacy. Excellent school libraries are essential if young people are to have access to the reading resources to help them gain the level of literacy achievement vital to meeting the challenge of the twenty-first century. Sections of the booklet…

  1. Library Connection Development.

    ERIC Educational Resources Information Center

    Berry, John N., III

    1990-01-01

    The John Wiley and Sons publishing company has made a commitment to publishing more tradebooks for the retail and library marketplace. As part of this expansion, Wiley has strengthened its library sales and promotion efforts and launched new publications and promotions to reach librarians. (MES)

  2. Branch Library Service.

    ERIC Educational Resources Information Center

    Perkins, John W.; And Others

    Designed for the training of a newly appointed branch librarian as well as for general background on the function of a branch library for the entire staff, this publication was written as a comprehensive guide to the administration of a branch library. Specific chapters focus on: (1) administrative goals and activities, (2) organizational…

  3. Library Technician Skill Standards.

    ERIC Educational Resources Information Center

    Highline Community Coll., Des Moines, WA.

    This document presents skill standards for library technicians. Introductory sections describe the industry and the job, what skill standards are, how the library technician skill standards were developed, employability skills and critical competencies, and the SCANS (Secretary's Commission on Achieving Necessary Skills) foundation skills profile.…

  4. Outreach Library Services.

    ERIC Educational Resources Information Center

    The Bookmark, 1985

    1985-01-01

    Focusing on the theme, Outreach Library Services, the articles in this publication provide a panoramic view of outreach services in eight of the public library systems in New York State. The compilation also contains four papers on special topics. Articles included are: (1) "Not So Trivial: In Pursuit of Outreach Services" (Joan F. Cooke, Finger…

  5. Library Censorship after Webster.

    ERIC Educational Resources Information Center

    Lee, Earl

    1989-01-01

    Discusses the potential of the Supreme Court's recent decision in Webster v. Reproductive Health Services, which upheld the Missouri anti-abortion statutes, to encourage state sponsored fiscal censorship in libraries. The types of library services and materials that might be affected are identified, and the First Amendment implications are…

  6. Academic Libraries in Japan

    ERIC Educational Resources Information Center

    Cullen, Rowena; Nagata, Haruki

    2008-01-01

    Academic libraries in Japan are well resourced by international standards, and support Japan's internationally recognized research capability well, but there are also ways in which they reflect Japan's strong bureaucratic culture. Recent changes to the status of national university libraries have seen a new interest in customer service, and…

  7. The Amazing Library Computer.

    ERIC Educational Resources Information Center

    Anderson, Eric S.

    1983-01-01

    Identifies computer software designed specifically for library management tasks in three distinct categories: library utility programs, and circulation and catalog programs for floppy disk and for hard disk. Company name and address, program, application, hardware, cost, and comments are provided in each category. (EJS)

  8. Planning the Electronic Library.

    ERIC Educational Resources Information Center

    Trolley, Jacqueline

    1995-01-01

    Introduces the Institute for Scientific Information's (ISI) electronic library project, and discusses the project's technological, economic, and legal issues. Describes computer system requirements that allow ISI's electronic library prototype to provide users with bibliographic information, abstracts, and full images of journals. (JMV)

  9. Insulin C-peptide test

    MedlinePlus

    C-peptide ... the test depends on the reason for the C-peptide measurement. Ask your health care provider if ... C-peptide is measured to tell the difference between insulin the body produces and insulin someone injects ...

  10. Constructing high complexity synthetic libraries of long ORFs using in vitro selection

    NASA Technical Reports Server (NTRS)

    Cho, G.; Keefe, A. D.; Liu, R.; Wilson, D. S.; Szostak, J. W.

    2000-01-01

    We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. Copyright 2000 Academic Press.

  11. The Charcot library.

    PubMed

    Philippon, Jacques H; Leroux-Hugon, Véronique M; Ricou, Philippe L; Poirier, Jacques G

    2003-02-01

    The Charcot library was officially created at the Salpêtrière Hospital in 1907 after the donation of the private library of Jean-Martin Charcot, which Dr. Charcot had accumulated progressively throughout his professional career. Increased by several other endowments (the most important being the collection of the Resident's Library, begun in 1886) and other private donations, the library became officially affiliated with the Paris VI University in 1985. It now functions as an entity oriented toward the neurosciences and offers a large number of 19th-century books (including all of Charcot's manuscripts) and more recent documents. Digitization of the most classic holdings, including atlases, will soon make them accessible through the library's web site.

  12. Fermilab Library projects

    SciTech Connect

    Garrett, P.; Ritchie, D.

    1990-05-03

    Preprint database management as done at various centers -- the subject of this workshop -- is hard to separate from the overall activities of the particular center. We therefore present the wider context at the Fermilab Library into which preprint database management fits. The day-to-day activities of the Library aside, the dominant activity at present is that of the ongoing Fermilab Library Automation. A less dominant but relatively time-consuming activity is that of doing more online searches in commercial databases on behalf of laboratory staff and visitors. A related activity is that of exploring the benefits of end-user searching of similar sources as opposed to library staff searching of the same. The Library Automation Project, which began about two years ago, is about to go fully online.'' The rationale behind this project is described in the documents developed during the December 1988--February 1989 planning phase.

  13. Mechanical force analysis of peptide interactions using atomic force microscopy.

    PubMed

    Nakamura, Chikashi; Takeda, Seiji; Kageshima, Masami; Ito, Miyuki; Sugimoto, Naoki; Sekizawa, Kazuko; Miyake, Jun

    2004-01-01

    Some peptides have previously been reported to bind low molecular weight chemicals. One such peptide with the amino acid sequence His-Ala-Ser-Tyr-Ser was selectively screened from a phage library and bound to a cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMpyP), with a binding constant of 10(5) M(-1) (J. Kawakami, T. Kitano, and N. Sugimoto, Chemical Communications, 1999, pp. 1765-1766). The proposed binding was due to pi-electron stacking from two aromatic amino acids of histidine and tyrosine. In this study, the weak interactions between TMpyP and the peptide were further investigated by force curve analysis using atomic force microscopy (AFM). The mechanical force required to unbind the peptide-porphyrin complex was measured by vertical movement of the AFM tip. Peptide self-assembled monolayers were formed on both a gold-coated mica substrate and a gold-coated AFM tip. The TMpyPs could bind between the two peptide layers when the peptide-immobilized AFM tip contacted the peptide-immobilized substrate in solution containing TMpyP. In the retracting process a force that ruptured the interaction between TMpyPs and peptides was observed. The unbinding force values correlated to the concentration of TMpyP. A detection limit of 100 ng/mL porphyrin was obtained for the force measurement, and was similar to surface plasmon resonance sensor detection limits. Furthermore, we calculated the product of the observed force and the length of the molecular elongation to determine the work required to unbind the complexes. The obtained values of unbinding work were in a reasonable range compared to the binding energy of porphyrin-peptide.

  14. Hydrazide Reactive Peptide Tags for Site-Specific Protein Labeling

    PubMed Central

    Eldridge, Glenn M.; Weiss, Gregory A.

    2011-01-01

    New site-specific protein labeling (SSPL) reactions for targeting specific, short peptides could be useful for the real time detection of proteins inside of living cells. One SSPL approach matches bioorthogonal reagents with complementary peptides. Here, hydrazide reactive peptides were selected from phage-displayed libraries using reaction-based selections. Selection conditions included washes of varying pH and treatment with NaCNBH3 in order to specifically select reactive carbonyl containing peptides. Selected peptides were fused to T4 lysozyme or synthesized on filter paper for colorimetric assays of the peptide-hydrazide interaction. A peptide-lysozyme protein fusion demonstrated specific, covalent labeling by the Hydrazide Reactive (HyRe) peptides in crude bacterial cell lysates, sufficient for the specific detection of an over-expressed protein fusion. Chemical synthesis of a short HyRe tag variant and subsequent reaction with two structurally distinct hydrazide probes produced covalent adducts observable by MALDI-TOF MS and MS/MS. Rather than isolating reactive carbonyl-containing peptides, we observed reaction with the N–terminal His of HyRe tag 114, amino acid sequence HKSNHSSKNRE, which attacks the hydrazide carbonyl at neutral pH. However, at the pH used during selection wash steps (<6.0), an alternative imine-containing product is formed that can be reduced with sodium cyanoborohydride. MSMS further reveals that this low pH product forms an adduct on Ser6. Further optimization of the novel bimolecular reaction described here could provide a useful tool for in vivo protein labeling and bioconjugate synthesis. The reported selection and screening methods could be widely applicable to the identification of peptides capable of other site-specific protein labeling reactions with bioorthogonal reagents. PMID:21905743

  15. Lest We Forget: Prison Libraries.

    ERIC Educational Resources Information Center

    Koons, Phil

    1988-01-01

    This discussion of prison library programs in Ohio covers: (1) financial support; (2) the nature of prison library work; (3) a typical day in the library; and (4) rewards of prison librarianship. (1 reference) (MES)

  16. Library Legislation: Some General Considerations

    ERIC Educational Resources Information Center

    Ladenson, Alex

    1970-01-01

    Library service has become a concern of government at all levels with each having its specific role to play. This introductory statement to this issue of Library Trends" indicates the major substantive areas of library legislation. (Author/NH)

  17. On the Accuracy and Limits of Peptide Fragmentation Spectrum Prediction

    PubMed Central

    Li, Sujun; Arnold, Randy J.; Tang, Haixu; Radivojac, Predrag

    2011-01-01

    We estimated the reproducibility of tandem mass fragmentation spectra for the widely-used collision-induced dissociation (CID) instruments. Using the Pearson correlation coefficient as a measure of spectral similarity, we found that the within-experiment reproducibility of fragment ion intensities is very high (about 0.85). However, across different experiments and instrument types/setups, the correlation decreases by more than 15% (to about 0.70). We further investigated the accuracy of current predictors of peptide fragmentation spectra and found that they are more accurate than the ad-hoc models generally used by search engines (e.g. SEQUEST) and, surprisingly, approaching the empirical upper limit set by the average across-experiment spectral reproducibility (especially for charge +1 and charge +2 precursor ions). These results provide evidence that, in terms of accuracy of modeling, predicted peptide fragmentation spectra provide a viable alternative to spectral libraries for peptide identification, with a higher coverage of peptides and lower storage requirements. Furthermore, using five data sets of proteome digests by two different proteases, we find that PeptideART (a data-driven machine learning approach) is generally more accurate than MassAnalyzer (an approach based on a kinetic model for peptide fragmentation) in predicting fragmentation spectra, but that both models are significantly more accurate than the ad-hoc models. Availability: PeptideART is freely available at www.informatics.indiana.edu/predrag. PMID:21175207

  18. Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases.

    PubMed

    Klecker, Maria; Dissmeyer, Nico

    2016-01-01

    The automated SPOT (synthetic peptide arrays on membrane support technique) synthesis technology has entrenched as a rapid and robust method to generate peptide libraries on cellulose membrane supports. The synthesis method is based on conventional Fmoc chemistry building up peptides with free N-terminal amino acids starting at their cellulose-coupled C-termini. Several hundreds of peptide sequences can be assembled with this technique on one membrane comprising a strong binding potential due to high local peptide concentrations. Peptide orientation on SPOT membranes qualifies this array type for assaying substrate specificities of N-recognins, the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Pioneer studies described binding capability of mammalian and yeast enzymes depending on a peptide's N-terminus. SPOT arrays have been successfully used to describe substrate specificity of N-recognins which are the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Here, we describe the implementation of SPOT binding assays with focus on the identification of N-recognin substrates, applicable also for plant NERD enzymes. PMID:27424747

  19. Bacteriocin Inducer Peptides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin produc...

  20. Introduction to peptide synthesis.

    PubMed

    Stawikowski, Maciej; Fields, Gregg B

    2012-08-01

    A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar substitute aspartame to clinically used hormones such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues.