Phage display selection of peptides that target calcium-binding proteins.

PubMed

Vetter, Stefan W

2013-01-01

Phage display allows to rapidly identify peptide sequences with binding affinity towards target proteins, for example, calcium-binding proteins (CBPs). Phage technology allows screening of 10(9) or more independent peptide sequences and can identify CBP binding peptides within 2 weeks. Adjusting of screening conditions allows selecting CBPs binding peptides that are either calcium-dependent or independent. Obtained peptide sequences can be used to identify CBP target proteins based on sequence homology or to quickly obtain peptide-based CBP inhibitors to modulate CBP-target interactions. The protocol described here uses a commercially available phage display library, in which random 12-mer peptides are displayed on filamentous M13 phages. The library was screened against the calcium-binding protein S100B.

99mTc-HYNIC-TNF analogs (WH701) derived from phage display peptide libraries for imaging TNF-receptor-positive ovarian carcinoma: preclinical evaluation

NASA Astrophysics Data System (ADS)

Xiang, Yan; Xia, Jinsong; Wu, H.; Li, H. F.

2002-04-01

Radiolabeled bioactive peptides which bind specifically to surface receptors over expressed in tumor cells are considered as alternatives for tumor detection with ECT. In this investigation, 99mTc-hydrazinonicotinyl - TNF analogs (WH701) was labeled using ethylenediaminediacetic acid (EDDA) as coligand (a number of TNF analogs had been selected and synthesized using random phage-display peptides library in our lab) and Pharmacokinetics and feasibility studies were performed.

Improving short antimicrobial peptides despite elusive rules for activity.

PubMed

Mikut, Ralf; Ruden, Serge; Reischl, Markus; Breitling, Frank; Volkmer, Rudolf; Hilpert, Kai

2016-05-01

Antimicrobial peptides (AMPs) can effectively kill a broad range of life threatening multidrug-resistant bacteria, a serious threat to public health worldwide. However, despite great hopes novel drugs based on AMPs are still rare. To accelerate drug development we studied different approaches to improve the antibacterial activity of short antimicrobial peptides. Short antimicrobial peptides seem to be ideal drug candidates since they can be synthesized quickly and easily, modified and optimized. In addition, manufacturing a short peptide drug will be more cost efficient than long and structured ones. In contrast to longer and structured peptides short AMPs seem hard to design and predict. Here, we designed, synthesized and screened five different peptide libraries, each consisting of 600 9-mer peptides, against Pseudomonas aeruginosa. Each library is presenting a different approach to investigate effectiveness of an optimization strategy. The data for the 3000 peptides were analyzed using models based on fuzzy logic bioinformatics and plausible descriptors. The rate of active or superior active peptides was improved from 31.0% in a semi-random library from a previous study to 97.8% in the best new designed library. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert. Copyright © 2015 Elsevier B.V. All rights reserved.

Ligand-regulated peptide aptamers.

PubMed

Miller, Russell A

2009-01-01

The peptide aptamer approach employs high-throughput selection to identify members of a randomized peptide library displayed from a scaffold protein by virtue of their interaction with a target molecule. Extending this approach, we have developed a peptide aptamer scaffold protein that can impart small-molecule control over the aptamer-target interaction. This ligand-regulated peptide (LiRP) scaffold, consisting of the protein domains FKBP12, FRB, and GST, binds to the cell-permeable small-molecule rapamycin and the binding of this molecule can prevent the interaction of the randomizable linker region connecting FKBP12 with FRB. Here we present a detailed protocol for the creation of a peptide aptamer plasmid library, selection of peptide aptamers using the LiRP scaffold in a yeast two-hybrid system, and the screening of those peptide aptamers for a ligand-regulated interaction.

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

USDA-ARS?s Scientific Manuscript database

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 ...

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

Development of a bacteriophage displayed peptide library and biosensor

NASA Astrophysics Data System (ADS)

Chin, Robert C.; Salazar, Noe; Mayo, Michael W.; Villavicencio, Victor I.; Taylor, Richard B.; Chambers, James P.; Valdes, James J.

1996-04-01

A miniaturized, handheld biosensor for identification of hazardous biowarfare agents with high specificity is being developed. An innovative biological recognition system based on bacteriophage displayed peptide receptors will be utilized in conjunction with the miniature biosensor technology being developed. A bacteriophage library has been constructed to provide the artificial receptors. The library can contain millions of bacteriophage with randomly displayed peptide sequences in the phage outer protein coat which act as binding sites for the agents of interest. This library will be used to 'bio-pan' for phages that bind to a number of toxins and infectious agents and can, thus, provide an endless supply of low cost, reliable, specific, and stable artificial receptors. The biosensor instrument will utilize evanescent wave, planar waveguide, far-red dyes, diode laser and miniature circuit technologies for performance and portability.

Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with a Random Peptide-Displaying Adenovirus Library

PubMed Central

Yoshida, Kimiko; Goto, Naoko; Ohnami, Shumpei; Aoki, Kazunori

2012-01-01

The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:23029088

Phage display: concept, innovations, applications and future.

PubMed

Pande, Jyoti; Szewczyk, Magdalena M; Grover, Ashok K

2010-01-01

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field. Copyright © 2010 Elsevier Inc. All rights reserved.

Peptide ligands targeting integrin alpha3beta1 in non-small cell lung cancer.

PubMed

Lau, Derick; Guo, Linlang; Liu, Ruiwu; Marik, Jan; Lam, Kit

2006-06-01

Lung cancer is one of the most common cancers and is the leading cause of cancer death. We wish to identify peptide ligands for unique cell surface receptors of non-small lung cancer with the hope of developing these ligands as diagnostic and therapeutic agents. Using the method of 'one-bead one-peptide' combinatorial chemistry, a library of random cyclic octapeptides was synthesized on polystyrene beads. This library was used to screen for peptides that promoted attachment of lung adenocarcinoma cells employing a 'cell-growth-on-bead' assay. Consensus peptide sequences of cNGXGXXc were identified. These peptides promoted cell adhesion by targeting integrin alpha3beta1 over-expressed in non-small lung cancer cells. These peptide beads can be applied to capture cancer cells in malignant pleural fluid for purpose of diagnosis of lung cancer.

Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.

PubMed

Körbelin, J; Hunger, A; Alawi, M; Sieber, T; Binder, M; Trepel, M

2017-08-01

Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.

Generation of a novel artificial TrkB agonist, BM17d99, using T7 phage-displayed random peptide libraries.

PubMed

Ohnishi, Toshiyuki; Sakamoto, Kotaro; Asami-Odaka, Asano; Nakamura, Kimie; Shimizu, Ayako; Ito, Takashi; Asami, Taiji; Ohtaki, Tetsuya; Inooka, Hiroshi

2017-01-29

Tropomyosin receptor kinase B (TrkB) is a known receptor of brain-derived neurotrophic factor (BDNF). Because it plays a critical role in the regulation of neuronal development, maturation, survival, etc., TrkB is a good target for drugs against central nervous system diseases. In this study, we aimed to generate peptidic TrkB agonists by applying random peptide phage display technology. After the phage panning against recombinant Fc-fused TrkB (TrkB-Fc), agonistic phages were directly screened against TrkB-expressing HEK293 cells. Through subsequent screening of the first-hit BM17 peptide-derived focus library, we successfully obtained the BM17d99 peptide, which had no sequence similarity with BDNF but had TrkB-binding capacity. We then synthesized a dimeric BM17d99 analog peptide that could phosphorylate or activate TrkB by facilitating receptor homodimerization. Treatment of TrkB-expressing HEK293 cells with the dimeric BM17d99 analog peptide significantly induced the phosphorylation of TrkB, suggesting that homodimerization of TrkB was enhanced by the dimeric peptide. This report demonstrates that our approach is useful for the generation of artificial peptidic agonists of cell surface receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Screening and identification of RhD antigen mimic epitopes from a phage display random peptide library for the serodiagnosis of haemolytic disease of the foetus and newborn.

PubMed

Wang, Jiao; Song, Jingjing; Zhou, Shuimei; Fu, Yourong; Bailey, Jeffrey A; Shen, Changxin

2018-01-16

Identification of RhD antigen epitopes is a key component in understanding the pathogenesis of haemolytic disease of the foetus and newborn. Research has indicated that phage display libraries are useful tools for identifying novel mimic epitopes (mimotopes) which may help to determine antigen specificity. We selected the mimotopes of blood group RhD antigen by affinity panning a phage display library using monoclonal anti-D. After three rounds of biopanning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and then sent for sequencing and peptides synthesis. Next, competitive ELISA and erythrocyte haemagglutination inhibition tests were carried out to confirm the inhibitory activity of the synthetic peptide. To evaluate the diagnostic performance of the synthetic peptide, a diagnostic ELISA was examined. Fourteen of 35 phage clones that were chosen randomly from the titering plate were considered to be positive. Following DNA sequencing and translation, 11 phage clones were found to represent the same peptide - RMKMLMMLMRRK (P4) - whereas each of the other three clones represented a unique peptide. Through the competitive ELISA and erythrocyte haemagglutination inhibition tests, the peptide (P4) was verified to have the ability to mimic the RhD antigen. The diagnostic ELISA for P4 proved to be sensitive (82.61%) and specific (88.57%). This study reveals that the P4 peptide can mimic RhD antigen and paves the way for the development of promising targeted diagnostic and therapeutic platforms for haemolytic disease of the foetus and newborn.

Bacteriophages and medical oncology: targeted gene therapy of cancer.

PubMed

Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

2014-08-01

Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology.

The structure of a thermophilic kinase shapes fitness upon random circular permutation

PubMed Central

Jones, Alicia M.; Mehta, Manan M.; Thomas, Emily E.; Atkinson, Joshua T.; Segall-Shapiro, Thomas H.; Liu, Shirley; Silberg, Jonathan J.

2016-01-01

Proteins can be engineered for synthetic biology through circular permutation, a sequence rearrangement where native protein termini become linked and new termini are created elsewhere through backbone fission. However, it remains challenging to anticipate a protein’s functional tolerance to circular permutation. Here, we describe new transposons for creating libraries of randomly circularly permuted proteins that minimize peptide additions at their termini, and we use transposase mutagenesis to study the tolerance of a thermophilic adenylate kinase (AK) to circular permutation. We find that libraries expressing permuted AK with either short or long peptides amended to their N-terminus yield distinct sets of active variants and present evidence that this trend arises because permuted protein expression varies across libraries. Mapping all sites that tolerate backbone cleavage onto AK structure reveals that the largest contiguous regions of sequence that lack cleavage sites are proximal to the phosphotransfer site. A comparison of our results with a range of structure-derived parameters further showed that retention of function correlates to the strongest extent with the distance to the phosphotransfer site, amino acid variability in an AK family sequence alignment, and residue-level deviations in superimposed AK structures. Our work illustrates how permuted protein libraries can be created with minimal peptide additions using transposase mutagenesis, and they reveal a challenge of maintaining consistent expression across permuted variants in a library that minimizes peptide additions. Furthermore, these findings provide a basis for interpreting responses of thermophilic phosphotransferases to circular permutation by calibrating how different structure-derived parameters relate to retention of function in a cellular selection. PMID:26976658

The Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutation.

PubMed

Jones, Alicia M; Mehta, Manan M; Thomas, Emily E; Atkinson, Joshua T; Segall-Shapiro, Thomas H; Liu, Shirley; Silberg, Jonathan J

2016-05-20

Proteins can be engineered for synthetic biology through circular permutation, a sequence rearrangement in which native protein termini become linked and new termini are created elsewhere through backbone fission. However, it remains challenging to anticipate a protein's functional tolerance to circular permutation. Here, we describe new transposons for creating libraries of randomly circularly permuted proteins that minimize peptide additions at their termini, and we use transposase mutagenesis to study the tolerance of a thermophilic adenylate kinase (AK) to circular permutation. We find that libraries expressing permuted AKs with either short or long peptides amended to their N-terminus yield distinct sets of active variants and present evidence that this trend arises because permuted protein expression varies across libraries. Mapping all sites that tolerate backbone cleavage onto AK structure reveals that the largest contiguous regions of sequence that lack cleavage sites are proximal to the phosphotransfer site. A comparison of our results with a range of structure-derived parameters further showed that retention of function correlates to the strongest extent with the distance to the phosphotransfer site, amino acid variability in an AK family sequence alignment, and residue-level deviations in superimposed AK structures. Our work illustrates how permuted protein libraries can be created with minimal peptide additions using transposase mutagenesis, and it reveals a challenge of maintaining consistent expression across permuted variants in a library that minimizes peptide additions. Furthermore, these findings provide a basis for interpreting responses of thermophilic phosphotransferases to circular permutation by calibrating how different structure-derived parameters relate to retention of function in a cellular selection.

Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces.

PubMed

Hakami, Abdulrahim R; Ball, Jonathan K; Tarr, Alexander W

2015-09-01

Phage-displayed random peptide libraries are widely used for identifying peptide interactions with proteins and other substrates. Selection of peptide ligands involves iterative rounds of affinity enrichment. The binding properties of the selected phage clones are routinely tested using immunoassay after propagation to high titre in a bacterial host and precipitation using polyethylene glycol (PEG) and high salt concentration. These immunoassays can suffer from low sensitivity and high background signals. Polysorbate 20 (Tween(®) 20) is a non-ionic detergent commonly used in immunoassay washing buffers to reduce non-specific binding, and is also used as a blocking reagent. We have observed that Tween 20 enhances non-specific M13 library phage binding in a peptide-independent manner. Other non-ionic detergents were also found to promote significant, dose-dependent non-specific phage binding in ELISA. This effect was not observed for assays using phage concentrated by ultracentrifugation, suggesting that interactions occur between detergents and the PEG-precipitated phage, irrespective of the displayed peptide motif. This artefact may impact on successful affinity selection of peptides from phage-display libraries. We propose alternative methods for screening phage libraries for identifying binding interactions with target ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Phage display as a technology delivering on the promise of peptide drug discovery.

PubMed

Hamzeh-Mivehroud, Maryam; Alizadeh, Ali Akbar; Morris, Michael B; Church, W Bret; Dastmalchi, Siavoush

2013-12-01

Phage display represents an important approach in the development pipeline for producing peptides and peptidomimetics therapeutics. Using randomly generated DNA sequences and molecular biology techniques, large diverse peptide libraries can be displayed on the phage surface. The phage library can be incubated with a target of interest and the phage which bind can be isolated and sequenced to reveal the displayed peptides' primary structure. In this review, we focus on the 'mechanics' of the phage display process, whilst highlighting many diverse and subtle ways it has been used to further the drug-development process, including the potential for the phage particle itself to be used as a drug carrier targeted to a particular pathogen or cell type in the body. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification of cancer-specific motifs in mimotope profiles of serum antibody repertoire.

PubMed

Gerasimov, Ekaterina; Zelikovsky, Alex; Măndoiu, Ion; Ionov, Yurij

2017-06-07

For fighting cancer, earlier detection is crucial. Circulating auto-antibodies produced by the patient's own immune system after exposure to cancer proteins are promising bio-markers for the early detection of cancer. Since an antibody recognizes not the whole antigen but 4-7 critical amino acids within the antigenic determinant (epitope), the whole proteome can be represented by a random peptide phage display library. This opens the possibility to develop an early cancer detection test based on a set of peptide sequences identified by comparing cancer patients' and healthy donors' global peptide profiles of antibody specificities. Due to the enormously large number of peptide sequences contained in global peptide profiles generated by next generation sequencing, the large number of cancer and control sera is required to identify cancer-specific peptides with high degree of statistical significance. To decrease the number of peptides in profiles generated by nextgen sequencing without losing cancer-specific sequences we used for generation of profiles the phage library enriched by panning on the pool of cancer sera. To further decrease the complexity of profiles we used computational methods for transforming a list of peptides constituting the mimotope profiles to the list motifs formed by similar peptide sequences. We have shown that the amino-acid order is meaningful in mimotope motifs since they contain significantly more peptides than motifs among peptides where amino-acids are randomly permuted. Also the single sample motifs significantly differ from motifs in peptides drawn from multiple samples. Finally, multiple cancer-specific motifs have been identified.

Protease-Resistant Peptide Ligands from a Knottin Scaffold Library

PubMed Central

Getz, Jennifer A.; Rice, Jeffrey J.; Daugherty, Patrick S.

2011-01-01

Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 109 variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution, slow dissociation rates, and were able to inhibit thrombin’s enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a two hour incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications. PMID:21615106

Peptide mimotopes of complex carbohydrates in Salmonella enterica serovar typhi which react with both carbohydrate-specific monoclonal antibody and polyclonal sera from typhoid patients.

PubMed

Thong, Kwai-Lin; Tang, Swee-Seong; Tan, Wen-Siang; Devi, Shamala

2007-01-01

Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.

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.

Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions

PubMed Central

2015-01-01

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. PMID:25265085

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.

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

PubMed

Bentley, L; Fehrsen, J; Jordaan, F; Huismans, H; du Plessis, D H

2000-04-01

VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

Selection of High-Affinity Peptidic Serine Protease Inhibitors with Increased Binding Entropy from a Back-Flip Library of Peptide-Protease Fusions.

PubMed

Sørensen, Hans Peter; Xu, Peng; Jiang, Longguang; Kromann-Hansen, Tobias; Jensen, Knud J; Huang, Mingdong; Andreasen, Peter A

2015-09-25

We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of peptide sequences that target to the brain using in vivo phage display.

PubMed

Li, Jingwei; Zhang, Qizhi; Pang, Zhiqing; Wang, Yuchen; Liu, Qingfeng; Guo, Liangran; Jiang, Xinguo

2012-06-01

Phage display technology could provide a rapid means for the discovery of novel peptides. To find peptide ligands specific for the brain vascular receptors, we performed a modified phage display method. Phages were recovered from mice brain parenchyma after administrated with a random 7-mer peptide library intravenously. A longer circulation time was arranged according to the biodistributive brain/blood ratios of phage particles. Following sequential rounds of isolation, a number of phages were sequenced and a peptide sequence (CTSTSAPYC, denoted as PepC7) was identified. Clone 7-1, which encodes PepC7, exhibited translocation efficiency about 41-fold higher than the random library phage. Immunofluorescence analysis revealed that Clone 7-1 had a significant superiority on transport efficiency into the brain compared with native M13 phage. Clone 7-1 was inhibited from homing to the brain in a dose-dependent fashion when cyclic peptides of the same sequence were present in a competition assay. Interestingly, the linear peptide (ATSTSAPYA, Pep7) and a scrambled control peptide PepSC7 (CSPATSYTC) did not compete with the phage at the same tested concentration (0.2-200 pg). Labeled by Cy5.5, PepC7 exhibited significant brain-targeting capability in in vivo optical imaging analysis. The cyclic conformation of PepC7 formed by disulfide bond, and the correct structure itself play a critical role in maintaining the selectivity and affinity for the brain. In conclusion, PepC7 is a promising brain-target motif never been reported before and it could be applied to targeted drug delivery into the brain.

ORF phage display to identify cellular proteins with different functions.

PubMed

Li, Wei

2012-09-01

Open reading frame (ORF) phage display is a new branch of phage display aimed at improving its efficiency to identify cellular proteins with specific binding or functional activities. Despite the success of phage display with antibody libraries and random peptide libraries, phage display with cDNA libraries of cellular proteins identifies a high percentage of non-ORF clones encoding unnatural short peptides with minimal biological implications. This is mainly because of the uncontrollable reading frames of cellular proteins in conventional cDNA libraries. ORF phage display solves this problem by eliminating non-ORF clones to generate ORF cDNA libraries. Here I summarize the procedures of ORF phage display, discuss the factors influencing its efficiency, present examples of its versatile applications, and highlight evidence of its capability of identifying biologically relevant cellular proteins. ORF phage display coupled with different selection strategies is capable of delineating diverse functions of cellular proteins with unique advantages. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Identification of chondrocyte-binding peptides by phage display.

PubMed

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

2013-07-01

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. Copyright © 2013 Orthopaedic Research Society.

Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library.

PubMed

Houimel, Mehdi; Mazzucchelli, Luca

2013-01-01

Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for (125)I-hCCL11 binding to hCCR3 with IC(50) ranging from 3.5 to 9.7μM. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced peritoneal eosinophilia. The development of peptides inhibiting the interactions between hCCR3 and its chemokine ligands will facilitate the development of small peptides antagonists with the hope of ameliorating chronic inflammatory diseases in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

PubMed

Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

2018-04-25

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones. Both peptides, experimental and designed, may be potential candidates to be developed as useful diagnostic or therapeutic ligand molecules in gastric cancer research. Creative Commons Attribution License

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

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

Biomathematical Description of Synthetic Peptide Libraries

PubMed Central

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

Identification of the immunogenic epitopes of the whole venom component of the Hemiscorpius lepturus scorpion using the phage display peptide library.

PubMed

Jahdasani, Roghaye; Jamnani, Fatemeh Rahimi; Behdani, Mahdi; Habibi-Anbouhi, Mahdi; Yardehnavi, Najmeh; Shahbazzadeh, Delavar; Kazemi-Lomedasht, Fatemeh

2016-12-15

The venom of the Hemiscorpius lepturus scorpion contains mixtures of bioactive compounds that disturb biochemical and physiological functions of the victims. Hemiscorpius lepturus envenomation is recognized as a serious health concern in tropical regions. So far, there is no preventive procedure, and the main focus is on treatment of victims with an antiserum purified from hyper-immunized horses. Although antisera can neutralize the venom, they, in some cases, lead to anaphylactic shock and even death. Selection of peptides mimicking antigenic and immunogenic epitopes of toxins from random peptide libraries is a novel approach for the development of recombinant toxins and poly-epitopic vaccine. To achieve this aim, a phage display peptide library and three rounds of biopanning were performed on immobilized antibodies (IgGs) purified from the sera of hyper-immunized horses. The results show that the highest binding of the phage to immobilized horse antibodies occurred in the third round of biopanning. Over 125 individual clones carrying mimotopes of Hemiscorpius lepturus toxins were selected and subjected for sequencing. The sequencing results identified unique peptides mimicking the antigenic and immunogenic epitopes of Hemiscorpius lepturus toxins. The results of this study provide a basis for further studies and the development of a putative epitopic vaccine and a recombinant toxin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Specific ligands for classical swine fever virus screened from landscape phage display library.

PubMed

Yin, Long; Luo, Yuzi; Liang, Bo; Wang, Fei; Du, Min; Petrenko, Valery A; Qiu, Hua-Ji; Liu, Aihua

2014-09-01

Classical swine fever (CSF) is a devastating infectious disease caused by classical swine fever virus (CSFV). The screening of CSFV-specific ligands is of great significance for diagnosis and treatment of CSF. Affinity selection from random peptide libraries is an efficient approach to discover ligands with high stability and specificity. Here, we screened phage ligands for the CSFV E2 protein from f8/8 landscape phage display library by biopanning and obtained four phage clones specific for the E2 protein of CSFV. Viral blocking assays indicated that the phage clone displaying the octapeptide sequence DRATSSNA remarkably inhibited the CSFV replication in PK-15 cells at a titer of 10(10) transduction units, as evidenced by significantly decreased viral RNA copies and viral titers. The phage-displayed E2-binding peptides have the potential to be developed as antivirals for CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

Phage-display screening identifies LMP1-binding peptides targeting the C-terminus region of the EBV oncoprotein.

PubMed

Ammous-Boukhris, Nihel; Mosbah, Amor; Sahli, Emna; Ayadi, Wajdi; Hadhri-Guiga, Boutheina; Chérif, Ameur; Gargouri, Ali; Mokdad-Gargouri, Raja

2016-11-01

Latent membrane protein 1 (LMP1), a major oncoprotein of Epstein Barr Virus (EBV) is responsible for transforming B lymphocytes in vitro. LMP1 is overexpressed in several EBV-associated malignancies, and different approaches have been developed to reduce its level and accordingly its oncogenic function in tumor tissues. This study aimed to use phage display peptide library to obtain peptides which could specifically bind to the cytoplasmic region of LMP1 to prevent its interaction with signaling proteins. The LMP1 C-terminus region was produced in bacterial E. coli and used as target for the phage library panning. After 3 rounds, 20 phage clones were randomly selected and 8 showed high binding affinity to the recombinant C-terminus LMP1 protein. The most interesting candidates are the FO5 "QPTKDSSPPLRV" and NO4 "STTSPPAVPHNN" peptides since both bind the C-terminus LMP1 as showed by molecular docking. Furthermore, sequence alignment revealed that the FO5 peptide shared sequence similarity with the Death Receptor 4 which belongs to the tumor necrosis factor-related apoptosis-inducing receptor which plays key role in anti-tumor immunity. Copyright © 2016 Elsevier Inc. All rights reserved.

A specific RAGE-binding peptide biopanning from phage display random peptide library that ameliorates symptoms in amyloid β peptide-mediated neuronal disorder.

PubMed

Cai, Cuizan; Dai, Xiaoyong; Zhu, Yujie; Lian, Mengyang; Xiao, Fei; Dong, Fangyuan; Zhang, Qihao; Huang, Yadong; Zheng, Qing

2016-01-01

Alzheimer's disease (AD) is an age-related neurodegenerative disorder in which amyloid β (Aβ) peptide accumulates in the brain. The receptor for advanced glycation end product (RAGE) is a cellular binding site for Aβ peptide and mediates amyloid β-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a specific high-affinity RAGE inhibitor (APDTKTQ named RP-1) from a phage display library. RP-1 bound to RAGE and inhibited Aβ peptide-induced cellular stress in human neuroblastoma SH-SYSY cells in vitro. Three amino acids in RP-1 are identical to those in the Aβ peptide. RP-1 shows high homology to the 16-23 (KLVFFAED) regions in Aβ peptide and high-affinity RAGE. Functional analyses indicated that RP-1 significantly reduced the level of reactive oxygen species (ROS) and ROS products and that it enhanced catalase and glutathione peroxidase (GPx) activity. Furthermore, it inactivated caspase3 and caspase9 and inhibited the upregulation of RAGE, nuclear factor-κB (NF-κB), and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) protein expression. In addition, RP-1 activated the PI3K/AKT signaling pathway, inhibiting the interaction between Bax and Bcl-2. Our data suggest that RP-1 is a potent RAGE blocker that effectively controls the progression of Aβ peptide-mediated brain disorders and that it may have potential as a disease-modifying agent for AD.

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries*

PubMed Central

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

2016-01-01

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. PMID:27161445

Identification of cytidine-5-triphosphate synthase1-selective inhibitory peptide from random peptide library displayed on T7 phage.

PubMed

Sakamoto, Kotaro; Ishibashi, Yoshihiro; Adachi, Ryutaro; Matsumoto, Shin-Ichi; Oki, Hideyuki; Kamada, Yusuke; Sogabe, Satoshi; Zama, Yumi; Sakamoto, Jun-Ichi; Tani, Akiyoshi

2017-08-01

Cytidine triphosphate synthase 1 (CTPS1) is an enzyme expressed in activated lymphocytes that catalyzes the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP) with ATP-dependent amination, using either L-glutamine or ammonia as the nitrogen source. Since CTP plays an important role in DNA/RNA synthesis, phospholipid synthesis, and protein sialyation, CTPS1-inhibition is expected to control lymphocyte proliferation and size expansion in inflammatory diseases. In contrast, CTPS2, an isozyme of CTPS1 possessing 74% amino acid sequence homology, is expressed in normal lymphocytes. Thus, CTPS1-selective inhibition is important to avoid undesirable side effects. Here, we report the discovery of CTpep-3: Ac-FRLGLLKAFRRLF-OH from random peptide libraries displayed on T7 phage, which exhibited CTPS1-selective binding with a K D value of 210nM in SPR analysis and CTPS1-selective inhibition with an IC 50 value of 110nM in the enzyme assay. Furthermore, two fundamentally different approaches, enzyme inhibition assay and HDX-MS, provided the same conclusion that CTpep-3 acts by binding to the amidoligase (ALase) domain on CTPS1. To our knowledge, CTpep-3 is the first CTPS1-selective inhibitor. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of lanthanum-specific peptides for future recycling of rare earth elements from compact fluorescent lamps.

PubMed

Lederer, Franziska L; Curtis, Susan B; Bachmann, Stefanie; Dunbar, W Scott; MacGillivray, Ross T A

2017-05-01

As components of electronic scrap, rare earth minerals are an interesting but little used source of raw materials that are highly important for the recycling industry. Currently, there exists no cost-efficient technology to separate rare earth minerals from an electronic scrap mixture. In this study, phage surface display has been used as a key method to develop peptides with high specificity for particular inorganic targets in electronic scrap. Lanthanum phosphate doped with cerium and terbium as part of the fluorescent phosphors of spent compact fluorescent lamps (CFL) was used as a target material of economic interest to test the suitability of the phage display method to the separation of rare earth minerals. One random pVIII phage library was screened for peptide sequences that bind specifically to the fluorescent phosphor LaPO 4 :Ce 3+ ,Tb 3+ (LAP). The library contained at least 100 binding pVIII peptides per phage particle with a diversity of 1 × 10 9 different phage per library. After three rounds of enrichment, a phage clone containing the surface peptide loop RCQYPLCS was found to bind specifically to LAP. Specificity and affinity of the identified phage bound peptide was confirmed by using binding and competition assays, immunofluorescence assays, and zeta potential measurements. Binding and immunofluorescence assays identified the peptide's affinity for the fluorescent phosphor components CAT (CeMgAl 11 O 19 :Tb 3+ ) and BAM (BaMgAl 10 O 17 :Eu 2+ ). No affinity was found for other fluorescent phosphor components such as YOX (Y 2 O 3 :Eu 3+ ). The binding specificity of the RCQYPLCS peptide loop was improved 3-51-fold by using alanine scanning mutagenesis. The identification of peptides with high specificity and affinity for special components in the fluorescent phosphor in CFLs provides a potentially new strategic approach to rare earth recycling. Biotechnol. Bioeng. 2017;114: 1016-1024. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Tumor-targeting peptides from combinatorial libraries*

PubMed Central

Liu, Ruiwu; Li, Xiaocen; Xiao, Wenwu; Lam, Kit S.

2018-01-01

Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges infighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors. PMID:27210583

Identification of anti-CD98 antibody mimotopes for inducing antibodies with antitumor activity by mimotope immunization.

PubMed

Saito, Misa; Kondo, Masahiro; Ohshima, Motohiro; Deguchi, Kazuki; Hayashi, Hideki; Inoue, Kazuyuki; Tsuji, Daiki; Masuko, Takashi; Itoh, Kunihiko

2014-04-01

A mimotope is an antibody-epitope-mimicking peptide retrieved from a phage display random peptide library. Immunization with antitumor antibody-derived mimotopes is promising for inducing antitumor immunity in hosts. In this study, we isolated linear and constrained mimotopes from HBJ127, a tumor-suppressing anti-CD98 heavy chain mAb, and determined their abilities for induction of antitumor activity equal to that of the parent antibody. We detected elevated levels of antipeptide responses, but failed to detect reactivity against native CD98-expressing HeLa cells in sera of immunized mice. Phage display panning and selection of mimotope-immunized mouse spleen-derived antibody Fab library showed that HeLa cell-reactive Fabs were successfully retrieved from the library. This finding indicates that native antigen-reactive Fab clones represented an undetectable minor population in mimotope-induced antibody repertoire. Functional and structural analysis of retrieved Fab clones revealed that they were almost identical to the parent antibody. From these results, we confirmed that mimotope immunization was promising for retrieving antitumor antibodies equivalent to the parent antibody, although the co-administration of adjuvant compounds such as T-cell epitope peptides and Toll-like receptor 4 agonist peptides is likely to be necessary for inducing stronger antitumor immunity than mimotope injection alone. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Analysis of Qa-1bPeptide Binding Specificity and the Capacity of Cd94/Nkg2a to Discriminate between Qa-1–Peptide Complexes

PubMed Central

Kraft, Jennifer R.; Vance, Russell E.; Pohl, Jan; Martin, Amy M.; Raulet, David H.; Jensen, Peter E.

2000-01-01

The major histocompatibility complex class Ib protein, Qa-1b, serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1b peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1b. A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1b can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1b. Flow cytometry experiments with Qa-1b tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1b complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells. PMID:10974028

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.

Tumor-targeting peptides from combinatorial libraries.

PubMed

Liu, Ruiwu; Li, Xiaocen; Xiao, Wenwu; Lam, Kit S

2017-02-01

Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges in fighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors. Copyright © 2017. Published by Elsevier B.V.

Novel Synthetic Antimicrobial Peptides against Streptococcus mutans▿

PubMed Central

He, Jian; Eckert, Randal; Pharm, Thanh; Simanian, Maurice D.; Hu, Chuhong; Yarbrough, Daniel K.; Qi, Fengxia; Anderson, Maxwell H.; Shi, Wenyuan

2007-01-01

Streptococcus mutans, a common oral pathogen and the causative agent of dental caries, has persisted and even thrived on the tooth surface despite constant removal and eradication efforts. In this study, we generated a number of synthetic antimicrobial peptides against this bacterium via construction and screening of several structurally diverse peptide libraries where the hydrophobicity and charge within each library was varied incrementally in order to generate a collection of peptides with different biochemical characteristics. From these libraries, we identified multiple peptides with robust killing activity against S. mutans. To further improve their effectiveness, the most bactericidal peptides from each library were synthesized together as one molecule, in various combinations, with and without a flexible peptide linker between each antimicrobial region. Many of these “fusion” peptides had enhanced killing activities in comparison with those of the original nonconjoined molecules. The results presented here illustrate that small libraries of biochemically constrained peptides can be used to generate antimicrobial peptides against S. mutans, several of which may be likely candidates for the development of anticaries agents. PMID:17296741

Phage selection of peptide "microantibodies".

PubMed

Fujiwara, Daisuke; Fujii, Ikuo

2013-01-01

A bioactive peptide capable of inhibiting protein-protein interactions has the potential to be a molecular tool for biological studies and a therapeutic by disrupting aberrant interactions involved in diseases. We have developed combinatorial libraries of peptides with helix-loop-helix structure, from which the isolated peptides have the constrained structure to reduce entropy costs in binding, resulting in high binding affinities for target molecules. Previously, we designed a de novo peptide of helix-loop-helix structure that we termed a "microantibody." Using the microantibody as a library scaffold, we have constructed a phage-display library to successfully isolate molecular-targeting peptides against a cytokine receptor (granulocyte colony-stimulating factor receptor), a protein kinase (Aurora-A), and a ganglioside (GM1). Protocols in this article describe a general procedure for the library construction and the library screening.

HIV-1 V3 loop crown epitope-focused mimotope selection by patient serum from random phage display libraries: implications for the epitope structural features.

PubMed

Gazarian, Karlen G; Palacios-Rodríguez, Yadira; Gazarian, Tatiana G; Huerta, Leonor

2013-06-01

The crown region of the V3 loop in HIV-1 that contains the conserved amino acid sequence GPGR/G is known as the principal neutralizing determinant due to the extraordinary ability of antibodies to this region to neutralize the virus. To complement the existing peptide models of this epitope, we describe a family of 18 phage-displayed peptides, which include linear 12mer and constrained 7mer peptides that was selected by screening random libraries with serum from HIV-1 subtype B-infected patients. The 7mer constrained peptides presented two conserved amino acid sequences: PR-L in N-terminus and GPG in the C-terminus. On the basis of these peptides we propose a mimotope model of the V3 crown epitope in which the PR-L and GPG sequences represent the two known epitope binding sites. The GPG, has the same function as the V3 crown GPGR sequence but without the involvement of the "R" despite its being considered as the signature of the epitope in B-subtype viruses. The PR-L contains a proline not existing in the epitope that is postulated to induce kinks in the backbones of all peptides and create a spatial element mimicking the N-terminal conformationally variable binding site. Rabbit serum to these mimotopes recognized the V3 peptides and moderately decreased the fusion between HIV-1 Env- and CD4-expressing Jurkat cells. This study proposes the efficient generation by means of patient sera of V3 epitope mimics validated by interaction with the antibodies to contemporary viruses induced in patients. The serum antibody-selectable mimotopes are sources of novel information on the fine structure-function properties of HIV-1 principal neutralizing domain and candidate anti-HIV-1 immunogens. Copyright © 2012 Elsevier Ltd. All rights reserved.

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 present on the surface of the virus particle and can accept foreign sequence insertions without disruption of protein folding and viral particle assembly, and (2) on the encapsidation of nucleic acid sequences encoding both the VLP and the peptide it displays. The experiments described here are aimed at satisfying the first of these two requirements by engineering efficient peptide display at two different sites in MS2 coat protein. First, we evaluated the suitability of the N-terminus of MS2 coat for peptide insertions. It was observed that random N-terminal 10-mer fusions generally disrupted protein folding and VLP assembly, but by bracketing the foreign sequences with certain specific dipeptides, these defects could be suppressed. Next, the suitability of a coat protein surface loop for foreign sequence insertion was tested. Specifically, random sequence peptides were inserted into the N-terminal-most AB-loop of a coat protein single-chain dimer. Again we found that efficient display required the presence of appropriate dipeptides bracketing the peptide insertion. Finally, it was shown that an N-terminal fusion that tended to interfere specifically with capsid assembly could be efficiently incorporated into mosaic particles when co-expressed with wild-type coat protein.

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 .

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.

Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

PubMed

Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

2017-11-13

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

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 uncorrected data. The peptide library and detection efficiencies presented here may serve as a resource to facilitate studies in the epigenetics and proteomics fields. PMID:25000943

Mimicry of the immunodominant conformation-dependent antigenic site of hepatitis A virus by motifs selected from synthetic peptide libraries.

PubMed

Mattioli, S; Imberti, L; Stellini, R; Primi, D

1995-09-01

Hepatitis A virus (HAV) is a positive-strand RNA virus with a genome length of approximately 7,480 nucleotides. Although HAV morphogenesis is thought to be similar to that of poliovirus, the prototype picornavirus, the complete characterization of the antigenic structure of this virus remains elusive. All the available evidences, however, support the existence, on HAV virions and empty capsids, of an immunodominant neutralization antigenic site which is conformation dependent and whose structure involves residues of both VP1 and VP3 capsid proteins. This particular feature and the difficulty of obtaining high virus yield in tissue cultures make HAV an ideal target for developing synthetic peptides that simulate the structure of its main antigenic determinant. To this end we utilized, in the present work, the divide-couple-recombine approach to generate a random library composed of millions of different hexapeptides. This vast library was screened with a well-characterized anti-HAV monoclonal antibody. By this strategy we identified a peptide that reacted specifically with monoclonal and polyclonal anti-HAV antibodies and, in mice, induced a specific anti-virus immune response. Furthermore, the peptide could also be used in an enzyme-linked immunosorbent assay for revealing a primary immunoglobulin M immune response in sera of acutely infected human patients. Interestingly, no sequence homology was found between the identified peptide and the HAV capsid proteins VP1 and VP3. Collectively, these data represent an additional important paradigm of a mimotope capable of mimicking an antigenic determinant with unknown tertiary structure.

Discovery of GPX4 inhibitory peptides from random peptide T7 phage display and subsequent structural analysis.

PubMed

Sakamoto, Kotaro; Sogabe, Satoshi; Kamada, Yusuke; Matsumoto, Shin-Ichi; Kadotani, Akito; Sakamoto, Jun-Ichi; Tani, Akiyoshi

2017-01-08

The phospholipid hydroperoxidase glutathione peroxidase (GPX4) is an enzyme that reduces lipid hydroperoxides in lipid membranes. Recently, GPX4 has been investigated as a target molecule that induces iron-dependent cell death (ferroptosis) selectively in cancer cells that express mutant Ras. GPX4 inhibitors have the potential to become novel anti-cancer drugs. However, there are no druggable pockets for conventional small molecules on the molecular surface of GPX4. To generate GPX4 inhibitors, we examined the use of peptides as an alternative to small molecules. By screening peptide libraries displayed on T7 phages, and analyzing the X-ray crystal structures of the peptides, we successfully identified one peptide that binds to near Sec73 of catalytic site and two peptides that bind to another site on GPX4. To our knowledge, this is the first study reporting GPX4 inhibitory peptides and their structural information. Copyright © 2016 Elsevier Inc. All rights reserved.

Mimotope mapping as a complementary strategy to define allergen IgE-epitopes: peach Pru p 3 allergen as a model.

PubMed

Pacios, Luis F; Tordesillas, Leticia; Cuesta-Herranz, Javier; Compes, Esther; Sánchez-Monge, Rosa; Palacín, Arantxa; Salcedo, Gabriel; Díaz-Perales, Araceli

2008-04-01

Lipid transfer proteins (LTPs) are the major allergens of Rosaceae fruits in the Mediterranean area. Pru p 3, the LTP and major allergen of peach, is a suitable model for studying food allergy and amino acid sequences related with its IgE-binding capacity. In this work, we sought to map IgE mimotopes on the structure of Pru p 3, using the combination of a random peptide phage display library and a three-dimensional modelling approach. Pru p 3-specific IgE was purified from 2 different pools of sera from peach allergic patients grouped by symptoms (OAS-pool or SYS-pool), and used for screening of a random dodecapeptide phage display library. Positive clones were further confirmed by ELISA assays testing individual sera from each pool. Three-dimensional modelling allowed location of mimotopes based on analysis of electrostatic properties and solvent exposure of the Pru p 3 surface. Twenty-one phage clones were selected using Pru p 3-specific IgE, 9 of which were chosen using OAS-specific IgE while the other 12 were selected with systemic-specific IgE. Peptide alignments revealed consensus sequences for each pool: L37 R39 T40 P42 D43 R44 A46 P70 S76 P78 Y79 for OAS-IgE, and N35 N36 L37 R39 T40 D43 A46 S76 I77 P78 for systemic-IgE. These 2 consensus sequences were mapped on the same surface of Pru p 3, corresponding to the helix 2-loop-helix 3 region and part of the non-structured C-terminal coil. Thus, 2 relevant conformational IgE-binding regions of Pru p 3 were identified using a random peptide phage display library. Mimotopes can be used to study the interaction between allergens and IgE, and to accelerate the process to design new vaccines and new immunotherapy strategies.

Peptide Transduction-Based Therapies for Prostate Cancer

DTIC Science & Technology

2004-06-01

using an M13 peptide phage display library. Initial screening of the library for transduction of tumors in vivo has identified peptides able to...marker conjugates may have to be tested. (Months 6-12, Year 1) Progress: These experiments have been initiated. Task 4. An M13 peptide phage display ... phage 12 amino acid control peptide display library (New England Biolabs, Beverly, MA ) was used. Briefly, One nude mouse bearing a human tumor line

Protein Transduction Based Therapies for Breast Cancer

DTIC Science & Technology

2006-07-01

we also have developed a method for screening for tissue-targeted transduction peptides using an M13 peptide phage display library. Using this...Instead the focus was on the ability to identify a tumor specific peptide. Task 4. An M13 peptide phage display library will be used for...cancespecific tumor lines by screening a peptide phage display library both in cell culture as well as in nude micebearing xenografts. Initial results in

Stress-Driven Selection of Novel Phenotypes

NASA Technical Reports Server (NTRS)

Fox, George E.; Stepaov, Victor G.; Liu, Yamei

2011-01-01

A process has been developed that can confer novel properties, such as metal resistance, to a host bacterium. This same process can also be used to produce RNAs and peptides that have novel properties, such as the ability to bind particular compounds. It is inherent in the method that the peptide or RNA will behave as expected in the target organism. Plasmid-born mini-gene libraries coding for either a population of combinatorial peptides or stable, artificial RNAs carrying random inserts are produced. These libraries, which have no bias towards any biological function, are used to transform the organism of interest and to serve as an initial source of genetic variation for stress-driven evolution. The transformed bacteria are propagated under selective pressure in order to obtain variants with the desired properties. The process is highly distinct from in vitro methods because the variants are selected in the context of the cell while it is experiencing stress. Hence, the selected peptide or RNA will, by definition, work as expected in the target cell as the cell adapts to its presence during the selection process. Once the novel gene, which produces the sought phenotype, is obtained, it can be transferred to the main genome to increase the genetic stability in the organism. Alternatively, the cell line can be used to produce novel RNAs or peptides with selectable properties in large quantity for separate purposes. The system allows for easy, large-scale purification of the RNAs or peptide products. The process has been reduced to practice by imposing sub-inhibitory concentrations of NiCl2 on cells of the bacterium Escherichia coli that were transformed separately with the peptide library and RNA library. The evolved resistant clones were isolated, and sequences of the selected mini-gene variants were established. Clones resistant to NiCl2 were found to carry identical plasmid variants with a functional mini-gene that specifically conferred significant nickel tolerance on the host cells. Sequencing of the selected mini-gene revealed a propensity of the encoded peptide to bind transient metal ions. Expression of the mini-gene markedly improved growth parameters of the evolved clones at sub-inhibitory concentrations of NiCl2 while being slightly detrimental in the absence of stress. Similar results have been obtained with the RNA libraries. Overall, the results demonstrate a very natural outcome of the selection experiments in which the mini-genes were expected to be either successfully integrated into bacterial genetic networks, or rejected depending upon their effect on host fitness. This described approach can be useful as a laboratory model to study the dynamics of bacterial adaptive evolution on the molecular level. It can also provide a strategy for screening expressed DNA libraries in search of novel genes with desirable properties.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phipps, Mary Lisa; Lillo, Antoinetta M.; Shou, Yulin

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

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

DOE PAGES

Phipps, Mary Lisa; Lillo, Antoinetta M.; Shou, Yulin; ...

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

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. Copyright © 2015. Published by Elsevier Ltd.

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.

Identification and Characterization of Strychnine-Binding Peptides Using Phage-Display Screening.

PubMed

Zhang, Fang; Wang, Min; Qiu, Zheng; Wang, Xiao-Meng; Xu, Chun-Lei; Zhang, Xia

2017-01-01

In drug development, phage display is a high-throughput method for identifying the specific cellular targets of drugs. However, insoluble small chemicals remain intractable to this technique because of the difficulty of presenting molecules to phages without occupying or destroying the limited functional groups. In the present study, we selected Strychnine (Stry) as a model compounda and sought to develope an alternative in vitro biopanning strategy against insoluble suspension. A phage library displaying random sequences of fifteen peptides was employed to screen for interactions between Stry and its cellular selective binding peptides, which are of great value to have a complete understanding of the mechanism of Stry for its antitumor activity. After four rounds of biopanning, a selection of 100 binding clones was randomly picked and subjected to modified proliferation and diffusion assays to evaluate the binding affinity of the clones. Finally, eleven clones were identified as positive binders. The corresponding peptides were synthesized and detected for their binding activities using surface plasmon resonance imaging (SPRi). Our study provides a feasible scheme for confirming the interaction of chemical compounds and cellular binding peptides. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries.

PubMed

Hiemstra, H S; van Veelen, P A; Schloot, N C; Geluk, A; van Meijgaarden, K E; Willemen, S J; Leunissen, J A; Benckhuijsen, W E; Amons, R; de Vries, R R; Roep, B O; Ottenhoff, T H; Drijfhout, J W

1998-10-15

Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.

Libraries of Peptide Fragmentation Mass Spectra Database

National Institute of Standards and Technology Data Gateway

SRD 1C NIST Libraries of Peptide Fragmentation Mass Spectra Database (Web, free access)   The purpose of the library is to provide peptide reference data for laboratories employing mass spectrometry-based proteomics methods for protein analysis. Mass spectral libraries identify these compounds in a more sensitive and robust manner than alternative methods. These databases are freely available for testing and development of new applications.

Three polypeptides screened from phage display random peptide library may be the receptor polypeptide of Mycoplasma genitalium adhesion protein.

PubMed

Deng, Xiangying; Zhu, Youcong; Dai, Pei; Yu, Minjun; Chen, Liesong; Zhu, Cuiming; You, Xiaoxing; Li, Lingling; Zeng, Yanhua

2018-04-28

Mycoplasma genitalium adhesion protein (MgPa) is a major adhesin of M. genitalium, a human pathogen associated with a series of genitourinary tract diseases. MgPa plays a very important role in M. genitalium adhering to the host cells. However, the exact receptor peptides or proteins of MgPa are still poorly understood so far. Three polypeptides (V-H-W-D-F-R-Q-W-W-Q-P-S), (D-W-S-S-W-V -Y-R-D-P-Q-T) and (H-Y-I-D-F-R-W) were previously screened from a phage display random peptide library using recombinant MgPa (rMgPa) as a target molecule. In this study, three polypeptides were artificially synthesized and investigated as to whether they are potential receptors of MgPa. We found that rMgPa specifically bound to three synthesized polypeptides as determined via an indirect enzyme-linked immunosorbent assay (ELISA). Moreover, three polypeptides were further identified by indirect immunofluorescence microscopy (IFM). We confirmed that rMgPa and M. genitalium can adhere to SV-HUC-1 cells in vitro and that anti-rMgPa antibody and three synthesized polypeptides can partially inhibit the adherence of rMgPa and M. genitalium to SV-HUC-1 cells. In summary, these three polypeptides may be the essential receptor peptides of MgPa, and may aid in enhancing the understanding of biological function of MgPa and the possible pathogenic mechanism of M. genitalium. Copyright © 2018 Elsevier Ltd. All rights reserved.

Spectrum-to-Spectrum Searching Using a Proteome-wide Spectral Library*

PubMed Central

Yen, Chia-Yu; Houel, Stephane; Ahn, Natalie G.; Old, William M.

2011-01-01

The unambiguous assignment of tandem mass spectra (MS/MS) to peptide sequences remains a key unsolved problem in proteomics. Spectral library search strategies have emerged as a promising alternative for peptide identification, in which MS/MS spectra are directly compared against a reference library of confidently assigned spectra. Two problems relate to library size. First, reference spectral libraries are limited to rediscovery of previously identified peptides and are not applicable to new peptides, because of their incomplete coverage of the human proteome. Second, problems arise when searching a spectral library the size of the entire human proteome. We observed that traditional dot product scoring methods do not scale well with spectral library size, showing reduction in sensitivity when library size is increased. We show that this problem can be addressed by optimizing scoring metrics for spectrum-to-spectrum searches with large spectral libraries. MS/MS spectra for the 1.3 million predicted tryptic peptides in the human proteome are simulated using a kinetic fragmentation model (MassAnalyzer version2.1) to create a proteome-wide simulated spectral library. Searches of the simulated library increase MS/MS assignments by 24% compared with Mascot, when using probabilistic and rank based scoring methods. The proteome-wide coverage of the simulated library leads to 11% increase in unique peptide assignments, compared with parallel searches of a reference spectral library. Further improvement is attained when reference spectra and simulated spectra are combined into a hybrid spectral library, yielding 52% increased MS/MS assignments compared with Mascot searches. Our study demonstrates the advantages of using probabilistic and rank based scores to improve performance of spectrum-to-spectrum search strategies. PMID:21532008

Systematic identification of substrates for profiling of secreted proteases from Aspergillus species.

PubMed

Schaal, René; Kupfahl, Claudio; Buchheidt, Dieter; Neumaier, Michael; Findeisen, Peter

2007-11-01

Reliable and early diagnosis of life-threatening invasive mycoses in neutropenic patients caused by fungi of the Aspergillus species remains challenging because current clinical diagnostic tools lack in sensitivity and/or specificity. During invasive growth a variety of fungal proteases are secreted into the bloodstream and protease profiling with reporter peptides might improve diagnosis of invasive aspergillosis in serum specimens. To characterise the specific protease activity of Aspergillus fumigatus and Aspergillus niger we analyzed Aspergillus culture supernatants, human serum and the mixture of both. A systematic screening for optimised protease substrates was performed using a random peptide library consisting of 360 synthetic peptides featuring fluorescence resonance energy transfer (FRET). We could identify numerous peptides that are selectively cleaved by fungus-specific proteases. These reporter peptides might be feasible for future protease profiling of serum specimens to improve diagnosis and monitoring of invasive aspergillosis.

Rapid Generation of a Nanocrystal-Labeled Peptide Library for Specific Identification of the Bacterium Clostrium Botulinum

DOE Office of Scientific and Technical Information (OSTI.GOV)

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.

High-Throughput Method for Ranking the Affinity of Peptide Ligands Selected from Phage Display Libraries

PubMed Central

González-Techera, A.; Umpiérrez-Failache, M.; Cardozo, S.; Obal, G.; Pritsch, O.; Last, J. A.; Gee, S. J.; Hammock, B. D.; González-Sapienza, G.

2010-01-01

The use of phage display peptide libraries allows rapid isolation of peptide ligands for any target selector molecule. However, due to differences in peptide expression and the heterogeneity of the phage preparations, there is no easy way to compare the binding properties of the selected clones, which operates as a major “bottleneck” of the technology. Here, we present the development of a new type of library that allows rapid comparison of the relative affinity of the selected peptides in a high-throughput screening format. As a model system, a phage display peptide library constructed on a phagemid vector that contains the bacterial alkaline phosphatase gene (BAP) was selected with an antiherbicide antibody. Due to the intrinsic switching capacity of the library, the selected peptides were transferred “en masse” from the phage coat protein to BAP. This was coupled to an optimized affinity ELISA where normalized amounts of the peptide–BAP fusion allow direct comparison of the binding properties of hundreds of peptide ligands. The system was validated by plasmon surface resonance experiments using synthetic peptides, showing that the method discriminates among the affinities of the peptides within 3 orders of magnitude. In addition, the peptide–BAP protein can find direct application as a tracer reagent. PMID:18393454

Developing Novel Therapeutics Targeting Undifferentiated and Castration-Resistant Prostate Cancer Stem Cells

DTIC Science & Technology

2016-10-01

identify PCSC- specific homing peptides ; and 2) To perform unbiased drug library screening to identify novel PCSC-targeting chemicals. In the past...display library (PDL) screening in PSA-/lo PCa cells to identify PCSC- specific homing peptides ; and 2) To perform unbiased drug library screening to...Goals of the Project (SOW): Aim 1: To perform phage display library (PDL) screening in PSA-/lo PCa cells to identify PCSC- specific homing peptides

PECAN: Library Free Peptide Detection for Data-Independent Acquisition Tandem Mass Spectrometry Data

PubMed Central

Ting, Ying S.; Egertson, Jarrett D.; Bollinger, James G.; Searle, Brian C.; Payne, Samuel H.; Noble, William Stafford; MacCoss, Michael J.

2017-01-01

In mass spectrometry-based shogun proteomics, data-independent acquisition (DIA) is an emerging technique for unbiased and reproducible measurement of protein mixtures. Without targeting a specific precursor ion, DIA MS/MS spectra are often highly multiplexed, containing product ions from multiple co-fragmenting precursors. Thus, detecting peptides directly from DIA data is challenging; most DIA data analyses require spectral libraries. Here we present a new library-free, peptide-centric tool PECAN that detects peptides directly from DIA data. PECAN reports evidence of detection based on product ion scoring, enabling detection of low abundance analytes with poor precursor ion signal. We benchmarked PECAN with chromatographic peak picking accuracy and peptide detection capability. We further validated PECAN detection with data-dependent acquisition and targeted analyses. Last, we used PECAN to build a library from DIA data and to query sequence variants. Together, these results show that PECAN detects peptides robustly and accurately from DIA data without using a library. PMID:28783153

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.

In vitro selection using a dual RNA library that allows primerless selection

PubMed Central

Jarosch, Florian; Buchner, Klaus; Klussmann, Sven

2006-01-01

High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC50 of 4.5 nM at 37°C. PMID:16855281

PSBinder: A Web Service for Predicting Polystyrene Surface-Binding Peptides.

PubMed

Li, Ning; Kang, Juanjuan; Jiang, Lixu; He, Bifang; Lin, Hao; Huang, Jian

2017-01-01

Polystyrene surface-binding peptides (PSBPs) are useful as affinity tags to build a highly effective ELISA system. However, they are also a quite common type of target-unrelated peptides (TUPs) in the panning of phage-displayed random peptide library. As TUP, PSBP will mislead the analysis of panning results if not identified. Therefore, it is necessary to find a way to quickly and easily foretell if a peptide is likely to be a PSBP or not. In this paper, we describe PSBinder, a predictor based on SVM. To our knowledge, it is the first web server for predicting PSBP. The SVM model was built with the feature of optimized dipeptide composition and 87.02% (MCC = 0.74; AUC = 0.91) of peptides were correctly classified by fivefold cross-validation. PSBinder can be used to exclude highly possible PSBP from biopanning results or to find novel candidates for polystyrene affinity tags. Either way, it is valuable for biotechnology community.

Identification and characterization of a salivary-pellicle-binding peptide by phage display.

PubMed

Cukkemane, Nivedita; Bikker, Floris J; Nazmi, Kamran; Brand, Henk S; Veerman, Enno C I

2014-05-01

Dental biofilms are associated with oral diseases, making their control necessary. One way to control them is to prevent initial bacterial adherence to the salivary pellicle and thereby eventually decrease binding of late colonizing potential pathogens. The goal of this study was to generate a salivary-pellicle-binding peptide (SPBP) with antifouling activity towards primary colonizing bacteria. In order to achieve this goal we aimed to: (i) identify novel SPBPs by phage display; (ii) characterize the binding and antifouling properties of the selected SPBPs. A library of 2×10(9) phages displaying a random sequence of 12-mer peptides was used to identify peptides that bound selectively to the in vitro salivary pellicle. Three rounds of panning resulted in the selection of 10 pellicle-binding phages, each displaying a novel peptide sequence. The peptides were synthesized and their binding to the in vitro salivary pellicle was characterized in the presence and absence of calcium ions and Tween-20. The antifouling property of hydroxyapatite (HA) and saliva-coated HA discs treated with and without SPBPs were evaluated against Streptococcus gordonii. Ten unique SPBPs were identified using the phage display. One of these peptides, SPBP 10 (NSAAVRAYSPPS), exhibited significant binding to the in vitro salivary pellicle which was neither influenced by calcium ions, nor affected by up to 0.5% Tween-20. Its antifouling property against S. gordonii was significantly higher on the treated surfaces than on untreated surfaces. Use of the phage display library enabled us to find a specific SPBP with antifouling property towards S. gordonii. Copyright © 2014 Elsevier Ltd. All rights reserved.

Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

PubMed Central

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 (S a). The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of S a and use them to define the sequencing operator (S e q). Sequencing without any bias and errors is S e q = S a 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 (C E N), which describes elimination or statistically significant downsampling, of specific reads during the sequencing process. PMID:24416071

A Combinatorial H4 Tail Library to Explore the Histone Code

PubMed Central

Garske, Adam L.; Craciun, Gheorghe; Denu, John M.

2008-01-01

Histone modifications modulate chromatin structure and function. A posttranslational modification-randomized, combinatorial library based on the first twenty-one residues of histone H4 was designed for systematic examination of proteins that interpret a histone code. The 800-member library represented all permutations of most known modifications within the N-terminal tail of histone H4. To determine its utility in a protein-binding assay, the on-bead library was screened with an antibody directed against phosphoserine 1 of H4. Among the hits, 59/60 sequences were phosphorylated at S1, while 30/30 of those selected from the non-hits were unphosphorylated. A 512-member version of the library was then used to determine the binding specificity of the double tudor domain of hJMJD2A, a histone demethylase involved in transcriptional repression. Global linear least squares fitting of modifications from the identified peptides (40 hits and 34 non-hits) indicated that methylation of K20 was the primary determinant for binding, but that phosphorylation/acetylation on neighboring sites attenuated the interaction. To validate the on-bead screen, isothermal titration calorimetry was performed with thirteen H4 peptides. Dissociation constants ranged from 1 mM - 1μM and corroborated the screening results. The general approach should be useful for probing the specificity of any histone-binding protein. PMID:18616348

Peptide library synthesis on spectrally encoded beads for multiplexed protein/peptide bioassays

NASA Astrophysics Data System (ADS)

Nguyen, Huy Q.; Brower, Kara; Harink, Björn; Baxter, Brian; Thorn, Kurt S.; Fordyce, Polly M.

2017-02-01

Protein-peptide interactions are essential for cellular responses. Despite their importance, these interactions remain largely uncharacterized due to experimental challenges associated with their measurement. Current techniques (e.g. surface plasmon resonance, fluorescence polarization, and isothermal calorimetry) either require large amounts of purified material or direct fluorescent labeling, making high-throughput measurements laborious and expensive. In this report, we present a new technology for measuring antibody-peptide interactions in vitro that leverages spectrally encoded beads for biological multiplexing. Specific peptide sequences are synthesized directly on encoded beads with a 1:1 relationship between peptide sequence and embedded code, thereby making it possible to track many peptide sequences throughout the course of an experiment within a single small volume. We demonstrate the potential of these bead-bound peptide libraries by: (1) creating a set of 46 peptides composed of 3 commonly used epitope tags (myc, FLAG, and HA) and single amino-acid scanning mutants; (2) incubating with a mixture of fluorescently-labeled antimyc, anti-FLAG, and anti-HA antibodies; and (3) imaging these bead-bound libraries to simultaneously identify the embedded spectral code (and thus the sequence of the associated peptide) and quantify the amount of each antibody bound. To our knowledge, these data demonstrate the first customized peptide library synthesized directly on spectrally encoded beads. While the implementation of the technology provided here is a high-affinity antibody/protein interaction with a small code space, we believe this platform can be broadly applicable to any range of peptide screening applications, with the capability to multiplex into libraries of hundreds to thousands of peptides in a single assay.

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

PubMed

Ploss, Martin; Facey, Sandra J; Bruhn, Carina; Zemel, Limor; Hofmann, Kathrin; Stark, Robert W; Albert, Barbara; Hauer, Bernhard

2014-02-10

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. 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. This study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and to crystalline Ni3B nanoparticles. We think that the identified strong binding sequences described here could potentially serve for the utilisation of M13 phage as a viable alternative to other methods to create tailor-made boride composite materials or new catalytic surfaces by a biologically driven nano-assembly synthesis and structuring.

Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

PubMed

El Zoeiby, Ahmed; Sanschagrin, François; Darveau, André; Brisson, Jean-Robert; Levesque, Roger C

2003-03-01

The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemoto, Naoto, E-mail: nemoto@fms.saitama-u.ac.jp; Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005; Janusys Corporation, 508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844

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 bymore » 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.« less

Somatostatin displayed on filamentous phage as a receptor-specific agonist

PubMed Central

Rousch, Mat; Lutgerink, Jan T; Coote, James; de Bruïne, Adriaan; Arends, Jan-Willem; Hoogenboom, Hennie R

1998-01-01

In search of methods to identify bio-active ligands specific for G protein-coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage-based selection and functional screening method. First, methods for panning peptide phage on cells were established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into phage(mid) vectors and fusion to either pIII or pVIII viral coat proteins. Peptide displaying phage bound to a polyclonal anti-somatostatin serum, and, more importantly, to several somatostatin receptor subtypes (Sst) expressed on transfected CHO-K1 cells, in a pattern which was dependent on the used display method. Binding was competed with somatostatin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selections of phage libraries. Binding of somatostatin displaying phage to sst2 on a reporter cell line, in which binding of natural ligand reduces secretion of alkaline phosphatase (via a cyclic AMP responsive element sensitive promoter), proved that the phage particles act as receptor-specific agonists. Less than 100 phage particles per cell were required for this activity, which is approximately 1000 fold less than soluble somatostatin, suggesting that phage binding interferes with normal receptor desensitization and/or recycling. The combination of biopanning of phage libraries on cells with functional screening of phage particles for receptor triggering activity, may be used to select novel, bio-active ligands from phage libraries of random peptides, antibody fragments, or libraries based on the natural receptor ligand. PMID:9776337

A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site.

PubMed

Brammer, Leighanne A; Bolduc, Benjamin; Kass, Jessica L; Felice, Kristin M; Noren, Christopher J; Hall, Marilena Fitzsimons

2008-02-01

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.

A new strategy for the preparation of peptide-targeted technetium and rhenium radiopharmaceuticals. The automated solid-phase synthesis, characterization, labeling, and screening of a peptide-ligand library targeted at the formyl peptide receptor.

PubMed

Stephenson, Karin A; Banerjee, Sangeeta Ray; Sogbein, Oyebola O; Levadala, Murali K; McFarlane, Nicole; Boreham, Douglas R; Maresca, Kevin P; Babich, John W; Zubieta, Jon; Valliant, John F

2005-01-01

A new solid-phase synthetic methodology was developed that enables libraries of peptide-based Tc(I)/Re(I) radiopharmaceuticals to be prepared using a conventional automated peptide synthesizer. Through the use of a tridentate ligand derived from N-alpha-Fmoc-l-lysine, which we refer to as a single amino acid chelate (SAAC), a series of 12 novel bioconjugates [R-NH(CO)ZLF(SAAC)G, R = ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, benzyl; Z = Met, Nle] that are designed to target the formyl peptide receptor (FPR) were prepared. Construction of the library was carried out in a multiwell format on an Advanced ChemTech 348 peptide synthesizer where multi-milligram quantities of each peptide were isolated in high purity without HPLC purification. After characterization, the library components were screened for their affinity for the FPR receptor using flow cytometry where the K(d) values were found to be in the low micromolar range (0.5-3.0 microM). Compound 5j was subsequently labeled with (99m)Tc(I) and the product isolated in high radiochemical yield using a simple Sep-Pak purification procedure. The retention time of the labeled compound matched that of the fully characterized Re-analogue which was prepared through the use of the same solid-phase synthesis methodology that was used to construct the library. The work reported here is a rare example of a method by which libraries of peptide-ligand conjugates and their rhenium complexes can be prepared.

The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization.

PubMed

Dong, Qian; Liang, Yuxue; Yan, Xinjian; Markey, Sanford P; Mirokhin, Yuri A; Tchekhovskoi, Dmitrii V; Bukhari, Tallat H; Stein, Stephen E

2018-04-01

We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins.

The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization

PubMed Central

Dong, Qian; Liang, Yuxue; Yan, Xinjian; Markey, Sanford P.; Mirokhin, Yuri A.; Tchekhovskoi, Dmitrii V.; Bukhari, Tallat H.; Stein, Stephen E.

2018-01-01

ABSTRACT We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins. PMID:29425077

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays.

PubMed

Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

2016-11-19

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays

PubMed Central

Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

2016-01-01

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides. PMID:28952593

Identification of a cardiac specific protein transduction domain by in vivo biopanning using a M13 phage peptide display library in mice.

PubMed

Zahid, Maliha; Phillips, Brett E; Albers, Sean M; Giannoukakis, Nick; Watkins, Simon C; Robbins, Paul D

2010-08-17

A peptide able to transduce cardiac tissue specifically, delivering cargoes to the heart, would be of significant therapeutic potential for delivery of small molecules, proteins and nucleic acids. In order to identify peptide(s) able to transduce heart tissue, biopanning was performed in cell culture and in vivo with a M13 phage peptide display library. A cardiomyoblast cell line, H9C2, was incubated with a M13 phage 12 amino acid peptide display library. Internalized phage was recovered, amplified and then subjected to a total of three rounds of in vivo biopanning where infectious phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was greater than with a cationic transduction domain. Biopanning using a peptide phage display library identified a peptide able to transduce heart tissue in vivo efficiently and specifically. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart.

Screening of Peptide Libraries Against Protective Antigen of Bacillus Anthracis in a Disposable Microfluidic Cartridge

DTIC Science & Technology

2011-11-28

New Reprint Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge W911NF-09-D-0001...against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge Report Title ABSTRACT See attached. Screening of Peptide...Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge Joshua M. Kogot1, Yanting Zhang2, Stephen J. Moore3

Building high-quality assay libraries for targeted analysis of SWATH MS data.

PubMed

Schubert, Olga T; Gillet, Ludovic C; Collins, Ben C; Navarro, Pedro; Rosenberger, George; Wolski, Witold E; Lam, Henry; Amodei, Dario; Mallick, Parag; MacLean, Brendan; Aebersold, Ruedi

2015-03-01

Targeted proteomics by selected/multiple reaction monitoring (S/MRM) or, on a larger scale, by SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) typically relies on spectral reference libraries for peptide identification. Quality and coverage of these libraries are therefore of crucial importance for the performance of the methods. Here we present a detailed protocol that has been successfully used to build high-quality, extensive reference libraries supporting targeted proteomics by SWATH MS. We describe each step of the process, including data acquisition by discovery proteomics, assertion of peptide-spectrum matches (PSMs), generation of consensus spectra and compilation of MS coordinates that uniquely define each targeted peptide. Crucial steps such as false discovery rate (FDR) control, retention time normalization and handling of post-translationally modified peptides are detailed. Finally, we show how to use the library to extract SWATH data with the open-source software Skyline. The protocol takes 2-3 d to complete, depending on the extent of the library and the computational resources available.

PECAN: library-free peptide detection for data-independent acquisition tandem mass spectrometry data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ting, Ying S.; Egertson, Jarrett D.; Bollinger, James G.

Data-independent acquisition (DIA) is an emerging mass spectrometry (MS)-based technique for unbiased and reproducible measurement of protein mixtures. DIA tandem mass spectrometry spectra are often highly multiplexed, containing product ions from multiple cofragmenting precursors. Detecting peptides directly from DIA data is therefore challenging; most DIA data analyses require spectral libraries. Here we present PECECAN (http://pecan.maccosslab.org), a library-free, peptide-centric tool that robustly and accurately detects peptides directly from DIA data. PECECAN reports evidence of detection based on product ion scoring, which enables detection of low-abundance analytes with poor precursor ion signal. We demonstrate the chromatographic peak picking accuracy and peptide detectionmore » capability of PECECAN, and we further validate its detection with data-dependent acquisition and targeted analyses. Lastly, we used PECECAN to build a plasma proteome library from DIA data and to query known sequence variants.« less

Biomolecular Recognition of Semiconductors and Magnetic Materials to Assemble Nanoparticle Heterostructures

DTIC Science & Technology

2002-01-01

shown that engineered viruses can recognize specific semiconductor surfaces through the selection by combinatorial phage display method. These specific... phage display libraries. The screening method selected for binding affinity of a population of random peptides displayed as part of the pIII minor coat...shorter spacing than expected distance ( M13 phage length: 880 nm) corresponds to the length scale imposed by the phage which formed the tilted

Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

PubMed

Yee, Andrew; Tan, Fen-Lai; Ginsburg, David

2013-01-01

von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

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

2013-05-03

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/.

A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix-assisted laser desorption/ionisation mass spectrometry.

PubMed

Gurevich-Messina, Juan M; Giudicessi, Silvana L; Martínez-Ceron, María C; Acosta, Gerardo; Erra-Balsells, Rosa; Cascone, Osvaldo; Albericio, Fernando; Camperi, Silvia A

2015-01-01

Short cyclic peptides have a great interest in therapeutic, diagnostic and affinity chromatography applications. The screening of 'one-bead-one-peptide' combinatorial libraries combined with mass spectrometry (MS) is an excellent tool to find peptides with affinity for any target protein. The fragmentation patterns of cyclic peptides are quite more complex than those of their linear counterparts, and the elucidation of the resulting tandem mass spectra is rather more difficult. Here, we propose a simple protocol for combinatorial cyclic libraries synthesis and ring opening before MS analysis. In this strategy, 4-hydroxymethylbenzoic acid, which forms a benzyl ester with the first amino acid, was used as the linker. A glycolamidic ester group was incorporated after the combinatorial positions by adding glycolic acid. The library synthesis protocol consisted in the following: (i) incorporation of Fmoc-Asp[2-phenylisopropyl (OPp)]-OH to Ala-Gly-oxymethylbenzamide-ChemMatrix, (ii) synthesis of the combinatorial library, (iii) assembly of a glycolic acid, (iv) couple of an Ala residue in the N-terminal, (v) removal of OPp, (vi) peptide cyclisation through side chain Asp and N-Ala amino terminus and (vii) removal of side chain protecting groups. In order to simultaneously open the ring and release each peptide, benzyl and glycolamidic esters were cleaved with ammonia. Peptide sequences could be deduced from the tandem mass spectra of each single bead evaluated. The strategy herein proposed is suitable for the preparation of one-bead-one-cyclic depsipeptide libraries that can be easily open for its sequencing by matrix-assisted laser desorption/ionisation MS. It employs techniques and reagents frequently used in a broad range of laboratories without special expertise in organic synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

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 during the screening procedure, best inhibits L. major growth kinetics in vitro, and reduces cutaneous lesions in BALB/c mice, thus showing great promise in the development of new therapeutic molecules. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of the venom from the Australian scorpion Urodacus yaschenkoi: Molecular mass analysis of components, cDNA sequences and peptides with antimicrobial activity.

PubMed

Luna-Ramírez, Karen; Quintero-Hernández, Veronica; Vargas-Jaimes, Leonel; Batista, Cesar V F; Winkel, Kenneth D; Possani, Lourival D

2013-03-01

The Urodacidae scorpions are the most widely distributed of the four families in Australia and represent half of the species in the continent, yet their venoms remain largely unstudied. This communication reports the first results of a proteome analysis of the venom of the scorpion Urodacus yaschenkoi performed by mass fingerprinting, after high performance liquid chromatography (HPLC) separation. A total of 74 fractions were obtained by HPLC separation allowing the identification of approximately 274 different molecular masses with molecular weights varying from 287 to 43,437 Da. The most abundant peptides were those from 1 K Da and 4-5 K Da representing antimicrobial peptides and putative potassium channel toxins, respectively. Three such peptides were chemically synthesized and tested against Gram-positive and Gram-negative bacteria showing minimum inhibitory concentration in the low micromolar range, but with moderate hemolytic activity. It also reports a transcriptome analysis of the venom glands of the same scorpion species, undertaken by constructing a cDNA library and conducting random sequencing screening of the transcripts. From the resultant cDNA library 172 expressed sequence tags (ESTs) were analyzed. These transcripts were further clustered into 120 unique sequences (23 contigs and 97 singlets). The identified putative proteins can be assorted in several groups, such as those implicated in common cellular processes, putative neurotoxins and antimicrobial peptides. The scorpion U. yaschenkoi is not known to be dangerous to humans and its venom contains peptides similar to those of Opisthacanthus cayaporum (antibacterial), Scorpio maurus palmatus (maurocalcin), Opistophthalmus carinatus (opistoporines) and Hadrurus gerstchi (scorpine-like molecules), amongst others. Copyright © 2012 Elsevier Ltd. All rights reserved.

Use of synthetic peptide libraries for the H-2Kd binding motif identification.

PubMed

Quesnel, A; Casrouge, A; Kourilsky, P; Abastado, J P; Trudelle, Y

1995-01-01

To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott

2010-06-25

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the {alpha}-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the designmore » of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques - yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis - to elucidate specificity determinants for binding to Bcl-x{sub L} versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x{sub L} selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x{sub L}, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x{sub L}-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x{sub L} binders.« less

Determinants of BH3 binding specificity for Mcl-1 vs. Bcl-xL

PubMed Central

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott; Fire, Emiko; Grant, Robert A.; Keating, Amy E.

2010-01-01

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Pro-survival members of the family interact with pro-apoptotic BH3-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the pro-apoptotic proteins to a conserved hydrophobic groove on the pro-survival proteins. Native BH3-only proteins exhibit selectivity in binding pro-survival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work we used two complementary techniques, yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis, to elucidate specificity determinants for binding to Bcl-xL vs. Mcl-1, two prominent pro-survival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-xL selectively, or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1 selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-xL, Bcl-2, Bcl-w and Bfl-1, whereas Bcl-xL selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 vs. Bcl-xL binders. PMID:20363230

Specificity studies on Kallikrein-related peptidase 7 (KLK7) and effects of osmolytes and glycosaminoglycans on its peptidase activity.

PubMed

Oliveira, Juliana R; Bertolin, Thiago C; Andrade, Douglas; Oliveira, Lilian C G; Kondo, Marcia Y; Santos, Jorge A N; Blaber, Michael; Juliano, Luiz; Severino, Beatrice; Caliendo, Giuseppe; Santagada, Vincenzo; Juliano, Maria A

2015-01-01

KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F, Y or M, and its S1' and S2' subsites showed selectivity for hydrophilic amino acids, particularly R and K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz-MISLM(↓)KRPPGFSPF(↓)RSSRI-NH2 ((↓)indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-NLY(↓)RVE-Q-EDDnp is the best synthetic substrate so far described for KLK7 [kcat/Km=455 (mMs)(-1)] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (Ki=33mM) and is present in skin moisturizing factor (124mM). The KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed. Copyright © 2014. Published by Elsevier B.V.

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Specificity profiling of protein-binding domains using one-bead-one-compound Peptide libraries.

PubMed

Kunys, Andrew R; Lian, Wenlong; Pei, Dehua

2012-12-01

One-bead-one-compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need for any special equipment, and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains); subsequently, the specificity information can be used to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains, and the related data analysis. Curr. Protoc. Chem. Biol. 4:331-355 © 2012 by John Wiley & Sons, Inc.

Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display.

PubMed

Hacker, David E; Hoinka, Jan; Iqbal, Emil S; Przytycka, Teresa M; Hartman, Matthew C T

2017-03-17

Highly constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly constrained peptides are challenging to prepare. Here, we present a method which utilizes two robust, orthogonal chemical steps to create highly constrained bicyclic peptide libraries. This technology was optimized to be compatible with in vitro selections by mRNA display. We performed side-by-side monocyclic and bicyclic selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nanomolar affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance.

Recombinant phage probes for Listeria monocytogenes

NASA Astrophysics Data System (ADS)

Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

2007-10-01

Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

Definition of agonists and design of antagonists for alloreactive T cell clones using synthetic peptide libraries.

PubMed

de Koster, H S; Vermeulen, C J; Hiemstra, H S; Amons, R; Drijfhout, J W; Koning, F

1999-04-01

Alloreactive T cells form an important barrier for organ transplantation. To reduce the risk of rejection patients are given immunosuppressive drugs, which increase the chance of infection and the incidence of malignancies. It has been shown that a large proportion of alloreactive T cells specifically recognize peptides present in the groove of the allogeneic MHC molecule. This implies that it might be possible to modulate the alloresponse by peptides with antagonistic properties, thus preventing rejection without the side effects of general immunosuppression. Peptide antagonists can be designed on the basis of the original agonist, yet for alloreactive T cells these agonists are usually unknown. In this study we have used a dedicated synthetic peptide library to identify agonists for HLA-DR3-specific alloreactive T cell clones. Based on these agonists, altered peptide ligands (APL) were designed. Three APL could antagonize an alloreactive T cell clone in its response against the library-derived agonist as well as in its response against the original allodeterminant, HLA-DR3. This demonstrates that peptide libraries can be used to design antagonists for alloreactive T cells without knowledge about the nature of the actual allostimulatory peptide. Since the most potent agonists are selected, this strategy permits detection of potent antagonists. The results, however, also suggest that the degree of peptide dependency of alloreactive T cell clones may dictate whether a peptide antagonist can be found for such clones. Whether peptide antagonists will be valuable in the development of donor-patient-specific immunosuppression may therefore depend on the specificity of the in vivo-generated alloreactive T cells.

[Peptide phage display in biotechnology and biomedicine].

PubMed

Kuzmicheva, G A; Belyavskaya, V A

2016-07-01

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

Immobilized OBOC combinatorial bead array to facilitate multiplicative screening.

PubMed

Xiao, Wenwu; Bononi, Fernanda C; Townsend, Jared; Li, Yuanpei; Liu, Ruiwu; Lam, Kit S

2013-07-01

One-bead-one-compound (OBOC) combinatorial library screening has been broadly utilized for the last two decades to identify small molecules, peptides or peptidomimetics targeting variable screening probes such as cell surface receptors, bacteria, protein kinases, phosphatases, proteases etc. In previous screening methods, library beads were suspended in solution and screened against one single probe. Only the positive beads were tracked and isolated for additional screens and finally selected for chemical decoding. During this process, the remaining negative beads were not tracked and discarded. Here we report a novel bead immobilization method such that a bead library array can be conveniently prepared and screened in its entirety, sequentially many times with a series of distinct probes. This method not only allows us to increase the screening efficiency but also permits us to determine the binding profile of each and every library bead against a large number of target receptors. As proof of concept, we serially screened a random OBOC disulfide containing cyclic heptapeptide library with three water soluble dyes as model probes: malachite green, bromocresol purple and indigo carmine. This multiplicative screening approach resulted in a rapid determination of the binding profile of each and every bead respective to each of the three dyes. Beads that interacted with malachite green only, bromocresol purple only, or both indigo carmine and bromocresol purple were isolated, and their peptide sequences were determined with microsequencer. Ultimately, the novel OBOC multiplicative screening approach could play a key role in the enhancement of existing on-bead assays such as whole cell binding, bacteria binding, protein binding, posttranslational modifications etc. with increased efficiency, capacity, and specificity.

The mimic epitopes of Mycobacterium tuberculosis screened by phage display peptide library have serodiagnostic potential for tuberculosis.

PubMed

Wang, Li; Deng, Xiangying; Liu, Haican; Zhao, Lanhua; You, Xiaolong; Dai, Pei; Wan, Kanglin; Zeng, Yanhua

2016-11-01

Mycobacterium tuberculosis is an obligate pathogenic bacterial species in the family of Mycobacteriaceae and attracts excessive immune responses which cause pathology of the lungs in active tuberculosis. The lack of more sensitive and effective diagnosis reagents advocates a further recognition for the fast diagnostic and immunological measures for tuberculosis. Here, two 12-mer peptides with core sequences of SVSVGMKPSPRP (CS1) and TMGFTAPRFPHY (CS2) were screened from a phage display random peptide library using the purified mixed tuberculosis-positive serum as a target. Enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay verified that positive phages exhibited strong binding affinity to mixed tuberculosis-positive serum. BLAST analysis showed that the two sequences may be mimotopes of the Mycobacterium tuberculosis The diagnostic potential for two synthetic mimotope peptides CS1 and CS2 was evaluated using different panels of serum samples (n = 181) by ELISA, and the diagnostic parameters were calculated. CS1 and CS2 achieved sensitivity of 89.41% and 85.88%, and specificities were 90.63% and 87.50%, respectively. We hypothesized that the diagnostic based on CS1 and CS2 may become a promising strategy to enhance the detection of Mycobacterium tuberculosis infection due to higher specificity and sensitivity. Therefore, CS1 and CS2 may possess potentials to provide an experimental basis for the diagnosis of tuberculosis. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

PubMed Central

2015-01-01

Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP. Screening of the CVa2X and CCa2X libraries with Rattus norvegicus PFTase revealed reaction by many known recognition sequences as well as numerous unknown ones. Some of the latter occur in the genomes of bacteria and viruses and may be important for pathogenesis, suggesting new targets for therapeutic intervention. Screening of the CVa2X library with alkyne-functionalized isoprenoid substrates showed that those prepared from C10 or C15 precursors gave similar results, whereas the analogue synthesized from a C5 unit gave a different pattern of reactivity. Lastly, the substrate specificities of PFTases from three organisms (R. norvegicus, Saccharomyces cerevisiae, and Candida albicans) were compared using CVa2X libraries. R. norvegicus PFTase was found to share more peptide substrates with S. cerevisiae PFTase than with C. albicans PFTase. In general, this method is a highly efficient strategy for rapidly probing the specificity of this important enzyme. PMID:24841702

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

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. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Strategies for the construction and use of peptide and antibody libraries displayed on phages.

PubMed

Pini, Alessandro; Giuliani, Andrea; Ricci, Claudia; Runci, Ylenia; Bracci, Luisa

2004-12-01

Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery. A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning. The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure. This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide. The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity. Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use. Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries. Particular attention is paid to advanced strategies for the construction, preservation and panning.

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

PubMed Central

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

2012-01-01

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

Identification of Novel Growth Regulators in Plant Populations Expressing Random Peptides1[OPEN

PubMed Central

Bao, Zhilong; Clancy, Maureen A.

2017-01-01

The use of chemical genomics approaches allows the identification of small molecules that integrate into biological systems, thereby changing discrete processes that influence growth, development, or metabolism. Libraries of chemicals are applied to living systems, and changes in phenotype are observed, potentially leading to the identification of new growth regulators. This work describes an approach that is the nexus of chemical genomics and synthetic biology. Here, each plant in an extensive population synthesizes a unique small peptide arising from a transgene composed of a randomized nucleic acid sequence core flanked by translational start, stop, and cysteine-encoding (for disulfide cyclization) sequences. Ten and 16 amino acid sequences, bearing a core of six and 12 random amino acids, have been synthesized in Arabidopsis (Arabidopsis thaliana) plants. Populations were screened for phenotypes from the seedling stage through senescence. Dozens of phenotypes were observed in over 2,000 plants analyzed. Ten conspicuous phenotypes were verified through separate transformation and analysis of multiple independent lines. The results indicate that these populations contain sequences that often influence discrete aspects of plant biology. Novel peptides that affect photosynthesis, flowering, and red light response are described. The challenge now is to identify the mechanistic integrations of these peptides into biochemical processes. These populations serve as a new tool to identify small molecules that modulate discrete plant functions that could be produced later in transgenic plants or potentially applied exogenously to impart their effects. These findings could usher in a new generation of agricultural growth regulators, herbicides, or defense compounds. PMID:28807931

Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins.

PubMed

Valdivieso-Torres, Leonardo; Sarangi, Anindita; Whidby, Jillian; Marcotrigiano, Joseph; Roth, Monica J

2015-12-30

Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism are not understood. This study highlights the advantage of constrained peptides within receptor binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 envelopes that are essential for viral infection have been identified in this study and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F and W) and basic (K and H) residues critical for viral titer and suggest that AII, BV2, and CP internal cysteines bond together in distinct ways. Retargeted gammaretroviral particles have broad applications for therapeutic use. Although great advances have been achieved in identifying new Env-host cell receptor pairs, the rules for designing optimal Env libraries are still unclear. We have found that isolates with an additional pair of cysteines within the randomized region have the highest transduction efficiencies. This emphasizes the importance of considering cysteine pairs in the design of new libraries. Furthermore, our data clearly indicate that these cysteines are essential for viral infectivity by presenting essential residues to the host cell receptor. These studies facilitate the screening of Env libraries for functional entry into target cells, allowing the identification of novel gammaretroviral Envs targeting alternative host cell receptors for gene and protein delivery. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

[Establishment of systemic lupus erythematosus-like murine model with Sm mimotope].

PubMed

Xie, Hong-Fu; Feng, Hao; Zeng, Hai-Yan; Li, Ji; Shi, Wei; Yi, Mei; Wu, Bin

2007-04-01

To establish systemic lupus erythematosus (SLE) -like murine model by immunizing BALB/C mice with Sm mimotope. Sm mimotope was identified by screening a 12-mer random peptide library with monoclonal anti-Smith antibody. Sm mimotope was initially defined with sandwich ELISA, DNA sequencing, and deduced amino acid sequence; and BALB/C mice were subcutaneously injected with mixture phages clones. Sera Sm antibody, anti-double stranded DNA (dsDNA) antibody, and antinuclear antibody (ANA) of mice were detected using direct immunofluorescence; kidney histological changes were examined by HE staining. Five randomly selected peptides were sequenced and the amino acid sequences IR, SQ, and PP were detected in a higher frequency. High-titer IgG autoantibodies of dsDNA, Sm, and ANA in the sera of experiment group were detected by ELISA 28 days after having been immunized by Sm mimotope. Proteinuria was detected 33 days later; immune complex and nephritis were observed in kidney specimens. SLE-like murine model can be successfully induced by Sm phage mimotope.

Selection of peptides for binding semiconductor and magnetic materials for the purpose of organizing nanoscaled materials

NASA Astrophysics Data System (ADS)

Whaley, Sandra Renee

A peptide combinatorial approach, also known as phage display, was used to isolate peptides with the ability to bind semiconductor (GaAs, GaN, and InP) and magnetic (Fe2O3 and Fe3O4) materials. The commercially available combinatorial libraries contain randomized peptides either twelve (Ph.D-12(TM)) or seven (Ph.D-C7C(TM)) amino acids in length. The peptides are displayed on the pIII protein of M13 bacteriophage, which have been imaged by atomic force microscopy and transmission electron microscopy. After seven rounds of phage selection with a constrained seven amino acid sequence library (Ph.D-C7C(TM)), two sequences were isolated for binding Fe3O4 (MG-127 and MG-78). The haematite surface was screened with the same library and four unique sequences were isolated after six rounds of selection (HM-95, HM-101, HM-103, and HM-111). According to binding experiments (MG-78 v. MG-127 on Fe3O 4, MG-127 v. HM-95 on Fe3O4 and Fe2O 3, and MG-127 v. HM-95 on gamma-Fe2O3), the MG-127 clone had the highest affinity for iron oxide surfaces (magnetite, haematite, and maghemite) among the clones tested. The Fe3O 4 clone MG-127 displayed the ability to organize Fe3O 4 nanoparticles along bundles of phage. The synthetic peptide analog of this clone was used in the organization of nanoparticles onto the surface of latex beads. The surfaces of the III-V semiconductors were studied using x-ray photoelectron spectroscopy to determine their reactivity in the aqueous conditions used for phage selection. The GaN surface was shown to oxidize the least under these conditions, aiding in the ability to isolate a consensus amino acid sequence responsible for binding to this surface. The G1-3 clone isolated for binding the GaAs (100) surface displayed preferential binding to the GaAs (100) surface over Si (100), GaAs (111) A, GaAs (111) B, and AlGaAs. The synthetic peptide analog of the G12-3 clone was found to preferentially bind to GaAs (100) over either GaAs (111) surfaces or InP (100). This peptide was used to immobilize 10 nm gold particles onto the surface of GaAs within ten minutes. From these results we have shown that it is possible to isolate peptides with high affinities for binding technologically relevant materials, even those not found in nature. These peptides can be used for the organization of pre-formed nanoparticles in solution and on the surface of semiconductor materials.

Development of bacterial display peptides for use in biosensing applications

NASA Astrophysics Data System (ADS)

Stratis-Cullum, Dimitra N.; Kogot, Joshua M.; Sellers, Michael S.; Hurley, Margaret M.; Sarkes, Deborah A.; Pennington, Joseph M.; Val-Addo, Irene; Adams, Bryn L.; Warner, Candice R.; Carney, James P.; Brown, Rebecca L.; Pellegrino, Paul M.

2012-06-01

Recent advances in synthetic library engineering continue to show promise for the rapid production of reagent technology in response to biological threats. A synthetic library of peptide mutants built off a bacterial host offers a convenient means to link the peptide sequence, (i.e., identity of individual library members) with the desired molecular recognition traits, but also allows for a relatively simple protocol, amenable to automation. An improved understanding of the mechanisms of recognition and control of synthetic reagent isolation and evolution remain critical to success. In this paper, we describe our approach to development of peptide affinity reagents based on peptide bacterial display technology with improved control of binding interactions for stringent evolution of reagent candidates, and tailored performance capabilities. There are four key elements to the peptide affinity reagent program including: (1) the diverse bacterial library technology, (2) advanced reagent screening amenable to laboratory automation and control, (3) iterative characterization and feedback on both affinity and specificity of the molecular interactions, and (3) integrated multiscale computational prescreening of candidate peptide ligands including in silico prediction of improved binding performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be presented. Recent highlights of on cell vs. off-cell affinity behavior and correlation of the results with advanced docking simulations on the protein-peptide system(s) are included. The potential of this technology and approach to enable rapid development of a new affinity reagent with unprecedented speed (less than one week) would allow for rapid response to new and constantly emerging threats.

Quantitative synthesis of genetically encoded glycopeptide libraries displayed on M13 phage.

PubMed

Ng, Simon; Jafari, Mohammad R; Matochko, Wadim L; Derda, Ratmir

2012-09-21

Phage display is a powerful technology that enables the discovery of peptide ligands for many targets. Chemical modification of phage libraries have allowed the identification of ligands with properties not encountered in natural polypeptides. In this report, we demonstrated the synthesis of 2 × 10(8) genetically encoded glycopeptides from a commercially available phage-displayed peptide library (Ph.D.-7) in a two-step, one-pot reaction in <1.5 h. Unlike previous reports, we bypassed genetic engineering of phage. The glycan moiety was introduced via an oxime ligation following oxidation of an N-terminal Ser/Thr; these residues are present in the peptide libraries at 20-30% abundance. The construction of libraries was facilitated by simple characterization, which directly assessed the yield and regioselectivity of chemical reactions performed on phage. This quantification method also allowed facile yield determination of reactions in 10(9) distinct molecules. We envision that the methodology described herein will find broad application in the synthesis of custom chemically modified phage libraries.

Utilization of specific and non-specific peptide interactions with inorganic nanomaterials on the surface of bacteriophage M13: Methodologies towards phage supported bi-functional materials

NASA Astrophysics Data System (ADS)

Avery, Kendra Nicole

Many types of organisms create a variety of nano and micro scale materials from precursors available in their surrounding environments by a process called biomineralization. As scientists begin to understand how these organisms utilize specific and non-specific interactions with a variety of biopolymers such as chitin, peptides, proteins and nucleic acids with these precursors to create inorganic/organic composite materials, they have begun to wonder about the synthesis of other types of non-biologically templated synthetic techniques that might be possible. Bioengineered organisms and biopolymers have begun to be used for these types of studies. A variety of selection techniques exist for discovering biopolymers with an affinity for a target material, however, one of the most notable is a technique called peptide phage display. This is a technique that utilizes a commercially available randomized peptide library attached at the tip of the filamentous bacteriophage M13. In this dissertation capabilities of bacteriophage M13 are explored in regard to the creation of bi-functional nano materials by exploiting both specific peptide interactions as well as non-specific peptide interactions on the surface of the organism. Chapter 2 focuses on utilizing the specific peptide interactions of the randomized library at pIII in order to discover peptides with high binding affinity for a variety of nanomaterials. Selection studies called biopanning are performed on a variety of nanomaterials such as CaMoO4, allotropes of Ni, Fe2O3 and Fe3O4, and Rh and Pt with the fcc type crystal structure. Similarities and differences between peptides discovered for these materials are discussed. Chapter 3 focuses on utilizing the non-specific peptide interactions on the long axis of M13 called pVIII. The pVIII region consists of 2700 copies of the same 50 amino acid protein which as a negatively charged domain which is exposed to solution. The pVIII region therefore provides the surface of the phage with a negative charge on which nanomaterials can be supported. Metal salt precursors reduced in the presence of WT M13 are studied in this chapter. Metal salt precursors of Fe, Co, Ru, Rh and Pd seem to be the most effective at coating the surface of the phage based on the positively charged metal-aquo complexes formed in water, which are attracted to the negative pVIII region. Other types of reactions are explored with WT phage as a scaffold such as conversion chemistry in a polyol solvent to access several intermetallic phases as well as co-precipitation reactions to access ternary oxides. Chapter 4 focuses on combining research from chapter 2 and chapter 3 to create a bi-functional material that utilizes both specific and non-specific peptide interactions with inorganic materials on the surface of M13 to attach two different types of nanomaterials. The example provided here is a magnetically recoverable hydrogenation catalyst made up of a pVIII region coated with rhodium nanoparticles held in place by non-specific peptide interactions and a pIII region attached to iron oxide nanoparticles via specific peptide interactions. This is the first example in the literature of a commercially available pIII bioengineered M13 bacteriophage forming a bi-functional material. This research provides a methodology to design and build single and multi-component materials on the surface of bacteriophage M13 without the necessity for additional bioengineering and library characterization. The simplicity of use will make the technique available to a wider variety of researchers in the materials science community.

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/. © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

PubMed

Alban, Silvana M; de Moura, Juliana Ferreira; Minozzo, João Carlos; Mira, Marcelo Távora; Soccol, Vanete Thomaz

2013-01-25

An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

PubMed Central

Edwards, W. Barry

2013-01-01

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy. PMID:23935860

Quantitation of the phosphoproteome using the library-assisted extracted ion chromatogram (LAXIC) strategy.

PubMed

Arrington, Justine V; Xue, Liang; Tao, W Andy

2014-01-01

Phosphorylation is a key posttranslational modification that regulates many signaling pathways, but quantifying changes in phosphorylation between samples can be challenging due to its low stoichiometry within cells. We have introduced a mass spectrometry-based label-free quantitation strategy termed LAXIC for the analysis of the phosphoproteome. This method uses a spiked-in synthetic peptide library designed to elute across the entire chromatogram for local normalization of phosphopeptides within complex samples. Normalization of phosphopeptides by library peptides that co-elute within a small time frame accounts for fluctuating ion suppression effects, allowing more accurate quantitation even when LC-MS performance varies. Here we explain the premise of LAXIC, the design of a suitable peptide library, and how the LAXIC algorithm can be implemented with software developed in-house.

Identification and immunogenicity of immunodominant mimotopes of outer membrane protein U (OmpU) of Vibrio mimicus from phage display peptide library.

PubMed

Cen, Junyu; Liu, Xueqin; Li, Jinnian; Zhang, Ming; Wang, Wei

2013-01-01

Vibrio mimicus (V. mimicus) is the causative agent of ascites disease in aquatic animals. Outer membrane protein U (OmpU) is an important antigen of V. mimicus, but its protective epitopes are still unclear. A random 12-mer phage-displayed peptide library was used to screen and identify immunodominant mimotopes of the OmpU protein in V. mimicus by panning against purified OmpU-specific polyclonal antibody. Then the immunogenicity and immunoprotection in fish of these mimotopes was evaluated. Nine positive phage clones presented seven different 12- peptide sequences and more than 50% of them carried a consensus core motif of DSSK-P. These positive clones reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by OmpU protein. Intraperitoneal injection of seven positive phage clones into fish induced a specific antibody response to OmpU protein. The fish immunized respectively with the positive phage clones C17, C24, C60 and C66 obtained 100% immunoprotective effect against experimental V. mimicus challenge. Taken together, these mimotopes presented by clone C17, C24, C60 and C66 were immunodominant mimotopes of the OmpU protein and exhibited a more appropriate candidate as epitope-based vaccine against V. mimicus infection in aquatic animals. Copyright © 2012 Elsevier Ltd. All rights reserved.

A novel FGFR3-binding peptide inhibits FGFR3 signaling and reverses the lethal phenotype of mice mimicking human thanatophoric dysplasia

PubMed Central

Jin, Min; Yu, Ying; Qi, Huabing; Xie, Yangli; Su, Nan; Wang, Xiaofeng; Tan, Qiaoyan; Luo, Fengtao; Zhu, Ying; Wang, Quan; Du, Xiaolan; Xian, Cory J.; Liu, Peng; Huang, Haiyang; Shen, Yue; Deng, Chu-Xia; Chen, Di; Chen, Lin

2012-01-01

Gain-of-function mutations in fibroblast growth factor receptor-3 (FGFR3) lead to several types of human skeletal dysplasia syndromes including achondroplasia, hypochondroplasia and thanatophoric dysplasia (TD). Currently, there are no effective treatments for these skeletal dysplasia diseases. In this study, we screened, using FGFR3 as a bait, a random 12-peptide phage library and obtained 23 positive clones that share identical amino acid sequences (VSPPLTLGQLLS), named as peptide P3. This peptide had high binding specificity to the extracellular domain of FGFR3. P3 inhibited tyrosine kinase activity of FGFR3 and its typical downstream molecules, extracellular signal-regulated kinase/mitogen-activated protein kinase. P3 also promoted proliferation and chondrogenic differentiation of cultured ATDC5 chondrogenic cells. In addition, P3 alleviated the bone growth retardation in bone rudiments from mice mimicking human thanatophoric dysplasia type II (TDII). Finally, P3 reversed the neonatal lethality of TDII mice. Thus, this study identifies a novel inhibitory peptide for FGFR3 signaling, which may serve as a potential therapeutic agent for the treatment of FGFR3-related skeletal dysplasia. PMID:23014564

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. Copyright © 2012 Elsevier B.V. All rights reserved.

Mass fingerprinting of the venom and transcriptome of venom gland of scorpion Centruroides tecomanus.

PubMed

Valdez-Velázquez, Laura L; Quintero-Hernández, Verónica; Romero-Gutiérrez, Maria Teresa; Coronas, Fredy I V; Possani, Lourival D

2013-01-01

Centruroides tecomanus is a Mexican scorpion endemic of the State of Colima, that causes human fatalities. This communication describes a proteome analysis obtained from milked venom and a transcriptome analysis from a cDNA library constructed from two pairs of venom glands of this scorpion. High perfomance liquid chromatography separation of soluble venom produced 80 fractions, from which at least 104 individual components were identified by mass spectrometry analysis, showing to contain molecular masses from 259 to 44,392 Da. Most of these components are within the expected molecular masses for Na(+)- and K(+)-channel specific toxic peptides, supporting the clinical findings of intoxication, when humans are stung by this scorpion. From the cDNA library 162 clones were randomly chosen, from which 130 sequences of good quality were identified and were clustered in 28 contigs containing, each, two or more expressed sequence tags (EST) and 49 singlets with only one EST. Deduced amino acid sequence analysis from 53% of the total ESTs showed that 81% (24 sequences) are similar to known toxic peptides that affect Na(+)-channel activity, and 19% (7 unique sequences) are similar to K(+)-channel especific toxins. Out of the 31 sequences, at least 8 peptides were confirmed by direct Edman degradation, using components isolated directly from the venom. The remaining 19%, 4%, 4%, 15% and 5% of the ESTs correspond respectively to proteins involved in cellular processes, antimicrobial peptides, venom components, proteins without defined function and sequences without similarity in databases. Among the cloned genes are those similar to metalloproteinases.

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.

Unique diversity of the venom peptides from the scorpion Androctonus bicolor revealed by transcriptomic and proteomic analysis.

PubMed

Zhang, Lei; Shi, Wanxia; Zeng, Xian-Chun; Ge, Feng; Yang, Mingkun; Nie, Yao; Bao, Aorigele; Wu, Shifen; E, Guoji

2015-10-14

Androctonus bicolor is one of the most poisonous scorpion species in the world. However, little has been known about the venom composition of the scorpion. To better understand the molecular diversity and medical significance of the venom from the scorpion, we systematically analyzed the venom components by combining transcriptomic and proteomic surveys. Random sequencing of 1000 clones from a cDNA library prepared from the venom glands of the scorpion revealed that 70% of the total transcripts code for venom peptide precursors. Our efforts led to a discovery of 103 novel putative venom peptides. These peptides include NaTx-like, KTx-like and CaTx-like peptides, putative antimicrobial peptides, defensin-like peptides, BPP-like peptides, BmKa2-like peptides, Kunitz-type toxins and some new-type venom peptides without disulfide bridges, as well as many new-type venom peptides that are cross-linked with one, two, three, five or six disulfide bridges, respectively. We also identified three peptides that are identical to known toxins from scorpions. The venom was also analyzed using a proteomic technique. The presence of a total of 16 different venom peptides was confirmed by LC-MS/MS analysis. The discovery of a wide range of new and new-type venom peptides highlights the unique diversity of the venom peptides from A. bicolor. These data also provide a series of novel templates for the development of therapeutic drugs for treating ion channel-associated diseases and infections caused by antibiotic-resistant pathogens, and offer molecular probes for the exploration of structures and functions of various ion channels. Copyright © 2015 Elsevier B.V. All rights reserved.

Application of plug-plug technique to ACE experiments for discovery of peptides binding to a larger target protein: a model study of calmodulin-binding fragments selected from a digested mixture of reduced BSA.

PubMed

Saito, Kazuki; Nakato, Mamiko; Mizuguchi, Takaaki; Wada, Shinji; Uchimura, Hiromasa; Kataoka, Hiroshi; Yokoyama, Shigeyuki; Hirota, Hiroshi; Kiso, Yoshiaki

2014-03-01

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a "plug-plug" technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan-X, β-endorphin, and oxytocin, as candidates for calmodulin-binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug-plug technique, the ACE-MS screening methodology successfully selected calmodulin-binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with Kd values between 8-147 μM for calmodulin were obtained from a Glu-C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE-MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

NIST Libraries of Peptide Fragmentation Mass Spectra Databass

National Institute of Standards and Technology Data Gateway

SRD 4 NIST Libraries of Peptide Fragmentation Mass Spectra Databass (PC database for purchase)   Interactive computer program for predicting thermodynamic and transport properties of pure fluids and fluid mixtures containing up to 20 components. The components are selected from a database of 196 components, mostly hydrocarbons.

Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions between clonotypic TCR and MHC peptide ligands.

PubMed

Zhao, Y; Gran, B; Pinilla, C; Markovic-Plese, S; Hemmer, B; Tzou, A; Whitney, L W; Biddison, W E; Martin, R; Simon, R

2001-08-15

The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.

Ligand design by a combinatorial approach based on modeling and experiment: application to HLA-DR4

NASA Astrophysics Data System (ADS)

Evensen, Erik; Joseph-McCarthy, Diane; Weiss, Gregory A.; Schreiber, Stuart L.; Karplus, Martin

2007-07-01

Combinatorial synthesis and large scale screening methods are being used increasingly in drug discovery, particularly for finding novel lead compounds. Although these "random" methods sample larger areas of chemical space than traditional synthetic approaches, only a relatively small percentage of all possible compounds are practically accessible. It is therefore helpful to select regions of chemical space that have greater likelihood of yielding useful leads. When three-dimensional structural data are available for the target molecule this can be achieved by applying structure-based computational design methods to focus the combinatorial library. This is advantageous over the standard usage of computational methods to design a small number of specific novel ligands, because here computation is employed as part of the combinatorial design process and so is required only to determine a propensity for binding of certain chemical moieties in regions of the target molecule. This paper describes the application of the Multiple Copy Simultaneous Search (MCSS) method, an active site mapping and de novo structure-based design tool, to design a focused combinatorial library for the class II MHC protein HLA-DR4. Methods for the synthesizing and screening the computationally designed library are presented; evidence is provided to show that binding was achieved. Although the structure of the protein-ligand complex could not be determined, experimental results including cross-exclusion of a known HLA-DR4 peptide ligand (HA) by a compound from the library. Computational model building suggest that at least one of the ligands designed and identified by the methods described binds in a mode similar to that of native peptides.

Solid-Phase Synthesis of Diverse Peptide Tertiary Amides By Reductive Amination

PubMed Central

Pels, Kevin; Kodadek, Thomas

2015-01-01

The synthesis of libraries of conformationally-constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as peptide tertiary amide (PTA). PTAs are strongly biased conformationally due to allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10,000 mixed peptoid-PTA oligomers. PMID:25695359

Solid-phase synthesis of diverse peptide tertiary amides by reductive amination.

PubMed

Pels, Kevin; Kodadek, Thomas

2015-03-09

The synthesis of libraries of conformationally constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as a peptide tertiary amide (PTA). PTAs are conformationally constrained because of allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10,000 mixed peptoid-PTA oligomers.

De novo designed library of linear helical peptides: an exploratory tool in the discovery of protein-protein interaction modulators.

PubMed

Bonache, M Ángeles; Balsera, Beatriz; López-Méndez, Blanca; Millet, Oscar; Brancaccio, Diego; Gómez-Monterrey, Isabel; Carotenuto, Alfonso; Pavone, Luigi M; Reille-Seroussi, Marie; Gagey-Eilstein, Nathalie; Vidal, Michel; de la Torre-Martinez, Roberto; Fernández-Carvajal, Asia; Ferrer-Montiel, Antonio; García-López, M Teresa; Martín-Martínez, Mercedes; de Vega, M Jesús Pérez; González-Muñiz, Rosario

2014-05-12

Protein-protein interactions (PPIs) have emerged as important targets for pharmaceutical intervention because of their essential role in numerous physiological and pathological processes, but screening efforts using small-molecules have led to very low hit rates. Linear peptides could represent a quick and effective approach to discover initial PPI hits, particularly if they have inherent ability to adopt specific peptide secondary structures. Here, we address this hypothesis through a linear helical peptide library, composed of four sublibraries, which was designed by theoretical predictions of helicity (Agadir software). The 13-mer peptides of this collection fixes either a combination of three aromatic or two aromatic and one aliphatic residues on one face of the helix (Ac-SSEEX(5)ARNX(9)AAX(12)N-NH2), since these are structural features quite common at PPIs interfaces. The 81 designed peptides were conveniently synthesized by parallel solid-phase methodologies, and the tendency of some representative library components to adopt the intended secondary structure was corroborated through CD and NMR experiments. As proof of concept in the search for PPI modulators, the usefulness of this library was verified on the widely studied p53-MDM2 interaction and on the communication between VEGF and its receptor Flt-1, two PPIs for which a hydrophobic α-helix is essential for the interaction. We have demonstrated here that, in both cases, selected peptides from the library, containing the right hydrophobic sequence of the hot-spot in one of the protein partners, are able to interact with the complementary protein. Moreover, we have discover some new, quite potent inhibitors of the VEGF-Flt-1 interaction, just by replacing one of the aromatic residues of the initial F(5)Y(9)Y(12) peptide by W, in agreement with previous results on related antiangiogenic peptides. Finally, the HTS evaluation of the full collection on thermoTRPs has led to a few antagonists of TRPV1 and TRPA1 channels, which open new avenues on the way to innovative modulators of these channels.

Enhancing the Breadth and Efficacy of Therapeutic Vaccines for Breast Cancer

DTIC Science & Technology

2014-10-01

us to generate full length recombinant TCR proteins that will be used to screen against the bacteriophage peptide library the Slansky/Kappler team has...the Denver team bacteriophage peptide library for HLA-A2 to screen these T cells. As a result we have abandoned this approach in order to focus our

Prospective identification of parasitic sequences in phage display screens

PubMed Central

Matochko, Wadim L.; Cory Li, S.; Tang, Sindy K.Y.; Derda, Ratmir

2014-01-01

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones. PMID:24217917

Stimulating CTL Towards HER2/neu Overexpressing Breast Cancer

DTIC Science & Technology

2000-10-01

cytotoxicity possibly due to the peptides poor solubility and poor binding affinity. To gain further insight into the factors that govern CTL activity, we...662 peptide. Objective: Complete by 12/96. Methods: A soluble recombinant form of HLA-A2 is folded in vitro in the presence of j2m and HN654-662. The...decided to substitute the first position from isoleucine to lysine to improve solubility of the peptide library. The library was used in our in vitro

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kay, B. K.; Castagnoli, L.; Biosciences Division

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.

Identification and characterization of mutant clones with enhanced propagation rates from phage-displayed peptide libraries.

PubMed

Nguyen, Kieu T H; Adamkiewicz, Marta A; Hebert, Lauren E; Zygiel, Emily M; Boyle, Holly R; Martone, Christina M; Meléndez-Ríos, Carola B; Noren, Karen A; Noren, Christopher J; Hall, Marilena Fitzsimons

2014-10-01

A target-unrelated peptide (TUP) can arise in phage display selection experiments as a result of a propagation advantage exhibited by the phage clone displaying the peptide. We previously characterized HAIYPRH, from the M13-based Ph.D.-7 phage display library, as a propagation-related TUP resulting from a G→A mutation in the Shine-Dalgarno sequence of gene II. This mutant was shown to propagate in Escherichia coli at a dramatically faster rate than phage bearing the wild-type Shine-Dalgarno sequence. We now report 27 additional fast-propagating clones displaying 24 different peptides and carrying 14 unique mutations. Most of these mutations are found either in or upstream of the gene II Shine-Dalgarno sequence, but still within the mRNA transcript of gene II. All 27 clones propagate at significantly higher rates than normal library phage, most within experimental error of wild-type M13 propagation, suggesting that mutations arise to compensate for the reduced virulence caused by the insertion of a lacZα cassette proximal to the replication origin of the phage used to construct the library. We also describe an efficient and convenient assay to diagnose propagation-related TUPS among peptide sequences selected by phage display. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures.

PubMed

Thomas, William D; Golomb, Miriam; Smith, George P

2010-12-15

Phage display is used to discover peptides or proteins with a desired target property-most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder's infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. Copyright © 2010 Elsevier Inc. All rights reserved.

Computational smart polymer design based on elastin protein mutability.

PubMed

Tarakanova, Anna; Huang, Wenwen; Weiss, Anthony S; Kaplan, David L; Buehler, Markus J

2017-05-01

Soluble elastin-like peptides (ELPs) can be engineered into a range of physical forms, from hydrogels and scaffolds to fibers and artificial tissues, finding numerous applications in medicine and engineering as "smart polymers". Elastin-like peptides are attractive candidates as a platform for novel biomaterial design because they exhibit a highly tunable response spectrum, with reversible phase transition capabilities. Here, we report the design of the first virtual library of elastin-like protein models using methods for enhanced sampling to study the effect of peptide chemistry, chain length, and salt concentration on the structural transitions of ELPs, exposing associated molecular mechanisms. We describe the behavior of the local molecular structure under increasing temperatures and the effect of peptide interactions with nearest hydration shell water molecules on peptide mobility and propensity to exhibit structural transitions. Shifts in the magnitude of structural transitions at the single-molecule scale are explained from the perspective of peptide-ion-water interactions in a library of four unique elastin-like peptide systems. Predictions of structural transitions are subsequently validated in experiment. This library is a valuable resource for recombinant protein design and synthesis as it elucidates mechanisms at the single-molecule level, paving a feedback path between simulation and experiment for smart material designs, with applications in biomedicine and diagnostic devices. Copyright © 2017. Published by Elsevier Ltd.

Mapping protease substrates using a biotinylated phage substrate library.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scholle, M. D.; Kriplani, U.; Pabon, A.

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-hydrophobicmore » is the scissile bond.« less

Novel ZnO-binding peptides obtained by the screening of a phage display peptide library

NASA Astrophysics Data System (ADS)

Golec, Piotr; Karczewska-Golec, Joanna; Łoś, Marcin; Węgrzyn, Grzegorz

2012-11-01

Zinc oxide (ZnO) is a semiconductor compound with a potential for wide use in various applications, including biomaterials and biosensors, particularly as nanoparticles (the size range of ZnO nanoparticles is from 2 to 100 nm, with an average of about 35 nm). Here, we report isolation of novel ZnO-binding peptides, by screening of a phage display library. Interestingly, amino acid sequences of the ZnO-binding peptides reported in this paper and those described previously are significantly different. This suggests that there is a high variability in sequences of peptides which can bind particular inorganic molecules, indicating that different approaches may lead to discovery of different peptides of generally the same activity (e.g., binding of ZnO) but having various detailed properties, perhaps crucial under specific conditions of different applications.

Spectra library assisted de novo peptide sequencing for HCD and ETD spectra pairs.

PubMed

Yan, Yan; Zhang, Kaizhong

2016-12-23

De novo peptide sequencing via tandem mass spectrometry (MS/MS) has been developed rapidly in recent years. With the use of spectra pairs from the same peptide under different fragmentation modes, performance of de novo sequencing is greatly improved. Currently, with large amount of spectra sequenced everyday, spectra libraries containing tens of thousands of annotated experimental MS/MS spectra become available. These libraries provide information of the spectra properties, thus have the potential to be used with de novo sequencing to improve its performance. In this study, an improved de novo sequencing method assisted with spectra library is proposed. It uses spectra libraries as training datasets and introduces significant scores of the features used in our previous de novo sequencing method for HCD and ETD spectra pairs. Two pairs of HCD and ETD spectral datasets were used to test the performance of the proposed method and our previous method. The results show that this proposed method achieves better sequencing accuracy with higher ranked correct sequences and less computational time. This paper proposed an advanced de novo sequencing method for HCD and ETD spectra pair and used information from spectra libraries and significant improved previous similar methods.

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.

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

PubMed

Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya; Yaguchi, Masahiro; Niida, Ayumu; Sasaki, Shigekazu; Miwa, Masanori; Ohkubo, Shoichi; Sakamoto, Jun-Ichi; Kamaura, Masahiro; Cho, Nobuo; Tani, Akiyoshi

2017-03-11

Amino-acid mutations of Gly 12 (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH 2 ) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. K D and IC 50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH 2 ) that inhibited enzyme activity of K-Ras(G12D) with IC 50  = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

Discovery of Neuropeptides in the Nematode Ascaris suum by Database Mining and Tandem Mass Spectrometry

PubMed Central

Jarecki, Jessica L.; Frey, Brian L.; Smith, Lloyd M.; Stretton, Antony O.

2011-01-01

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to discover peptides in extracts of the large parasitic nematode Ascaris suum. This required the assembly of a new database of known and predicted peptides. In addition to those already sequenced, peptides were either previously predicted to be processed from precursor proteins identified in an A. suum library of expressed sequence tags (ESTs), or newly predicted from a library of A. suum genome survey sequences (GSSs). The predicted MS/MS fragmentation patterns of this collection of real and putative peptides were compared with the actual fragmentation patterns found in the MS/MS spectra of peptides fractionated by MS; this enabled individual peptides to be sequenced. Many previously identified peptides were found, and 21 novel peptides were discovered. Thus, this approach is very useful, despite the fact that the available GSS database is still preliminary, having only 1X coverage. PMID:21524146

CiPerGenesis, A Mutagenesis Approach that Produces Small Libraries of Circularly Permuted Proteins Randomly Opened at a Focused Region: Testing on the Green Fluorescent Protein.

PubMed

Gaytán, Paul; Roldán-Salgado, Abigail; Yáñez, Jorge A; Morales-Arrieta, Sandra; Juárez-González, Víctor R

2018-06-12

Circularly permuted proteins (cpPs) represent a novel type of mutant proteins with original termini that are covalently linked through a peptide connector and opened at any other place of the polypeptide backbone to create new ends. cpPs are finding wide applications in biotechnology because their properties may be quite different from those of the parental protein. However, the actual challenge for the creation of successful cpPs is to identify those peptide bonds that can be broken to create new termini and ensure functional and well-folded cpPs. Herein, we describe CiPerGenesis, a combinatorial mutagenesis approach that uses two oligonucleotide libraries to amplify a circularized gene by PCR, starting and ending from a focused target region. This approach creates small libraries of circularly permuted genes that are easily cloned in the correct direction and frame using two different restriction sites encoded in the oligonucleotides. Once expressed, the protein libraries exhibit a unique sequence diversity, comprising cpPs that exhibit ordinary breakpoints between adjacent amino acids localized at the target region as well as cpPs with new termini containing user-defined truncations and repeats of some amino acids. CiPerGenesis was tested at the lid region G134-H148 of green fluorescent protein (GFP), revealing that the most fluorescent variants were those starting at Leu141 and ending at amino acids Tyr145, Tyr143, Glu142, Leu141, Lys140, and H139. Purification and biochemical characterization of some variants suggested a differential expression, solubility and maturation extent of the mutant proteins as the likely cause for the variability in fluorescence intensity observed in colonies.

Synthesis and screening of one-bead-one-compound cyclic peptide libraries.

PubMed

Qian, Ziqing; Upadhyaya, Punit; Pei, Dehua

2015-01-01

Cyclic peptides have been a rich source of biologically active molecules. Herein we present a method for the combinatorial synthesis and screening of large one-bead-one-compound (OBOC) libraries of cyclic peptides against biological targets such as proteins. Up to ten million different cyclic peptides are rapidly synthesized on TentaGel microbeads by the split-and-pool synthesis method and subjected to a multistage screening protocol which includes magnetic sorting, on-bead enzyme-linked and fluorescence-based assays, and in-solution binding analysis of cyclic peptides selectively released from single beads by fluorescence anisotropy. Finally, the most active hit(s) is identified by the partial Edman degradation-mass spectrometry (PED-MS) method. This method allows a single researcher to synthesize and screen up to ten million cyclic peptides and identify the most active ligand(s) in ~1 month, without the time-consuming and expensive hit resynthesis or the use of any special equipment.

Cell targeting peptides as smart ligands for targeting of therapeutic or diagnostic agents: a systematic review.

PubMed

Mousavizadeh, Ali; Jabbari, Ali; Akrami, Mohammad; Bardania, Hassan

2017-10-01

Cell targeting peptides (CTP) are small peptides which have high affinity and specificity to a cell or tissue targets. They are typically identified by using phage display and chemical synthetic peptide library methods. CTPs have attracted considerable attention as a new class of ligands to delivery specifically therapeutic and diagnostic agents, because of the fact they have several advantages including easy synthesis, smaller physical sizes, lower immunogenicity and cytotoxicity and their simple and better conjugation to nano-carriers and therapeutic or diagnostic agents compared to conventional antibodies. In this systematic review, we will focus on the basic concepts concerning the use of cell-targeting peptides (CTPs), following the approaches of selecting them from peptide libraries. We discuss several developed strategies for cell-specific delivery of different cargos by CTPs, which are designed for drug delivery and diagnostic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.

PubMed

Hu, Hongbo; Li, Li; Kao, Richard Y; Kou, Binbin; Wang, Zhanguo; Zhang, Liang; Zhang, Huiyuan; Hao, Zhiyong; Tsui, Wayne H; Ni, Anping; Cui, Lianxian; Fan, Baoxing; Guo, Feng; Rao, Shuan; Jiang, Chengyu; Li, Qian; Sun, Manji; He, Wei; Liu, Gang

2005-01-01

A 10-mer overlapping peptide library has been synthesized for screening and identification of linear B-cell epitopes of severe acute respiratory syndrome associated coronavirus (SARS-CoV), which spanned the major structural proteins of SARS-CoV. One hundred and eleven candidate peptides were positive according to the result of PEPscan, which were assembled into 22 longer peptides. Five of these peptides showed high cross-immunoreactivities (approximately 66.7 to 90.5%) to SARS convalescent patients' sera from the severest epidemic regions of the China mainland. Most interestingly, S(471-503), a peptide located at the receptor binding domain (RBD) of SARS-CoV, could specifically block the binding between the RBD and angiotensin-converting enzyme 2, resulting in the inhibition of SARS-CoV entrance into host cells in vitro. The study demonstrated that S(471-503) peptide was a potential immunoantigen for the development of peptide-based vaccine or a candidate for further drug evaluation against the SARS-CoV virus-cell fusion.

An antimicrobial bicyclic peptide from chemical space against multidrug resistant Gram-negative bacteria.

PubMed

Di Bonaventura, Ivan; Baeriswyl, Stéphane; Capecchi, Alice; Gan, Bee-Ha; Jin, Xian; Siriwardena, Thissa N; He, Runze; Köhler, Thilo; Pompilio, Arianna; Di Bonaventura, Giovanni; van Delden, Christian; Javor, Sacha; Reymond, Jean-Louis

2018-05-15

We used the concept of chemical space to explore a virtual library of bicyclic peptides formed by double thioether cyclization of a precursor linear peptide, and identified an antimicrobial bicyclic peptide (AMBP) with remarkable activity against several MDR strains of Acinetobacter baumannii and Pseudomonas aeruginosa.

Comprehensive computational design of ordered peptide macrocycles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hosseinzadeh, Parisa; Bhardwaj, Gaurav; Mulligan, Vikram Khipple

Mixed chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to-date, but there is currently no way to systematically search through the structural space spanned by such compounds for new drug candidates. Natural proteins do not provide a useful guide: peptide macrocycles lack regular secondary structures and hydrophobic cores and have different backbone torsional constraints. Hence the development of new peptide macrocycles has been approached by modifying natural products or using library selection methods; the former is limited by the small number of known structures, and the latter by the limited size and diversity accessible throughmore » library-based methods. To overcome these limitations, here we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L and D amino acids. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. We synthesize and characterize by NMR twelve 7-10 residue macrocycles, 9 of which have structures very close to the design models in solution. NMR structures of three 11-14 residue bicyclic designs are also very close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide based macrocycles unparalleled for other molecular systems, and vastly increase the available starting scaffolds for both rational drug design and library selection methods.« less

Phage display biopanning and isolation of target-unrelated peptides: in search of nonspecific binders hidden in a combinatorial library.

PubMed

Bakhshinejad, Babak; Zade, Hesam Motaleb; Shekarabi, Hosna Sadat Zahed; Neman, Sara

2016-12-01

Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in the discovery of numerous high-affinity target-binding peptides with potential application in drug discovery and delivery, the enrichment of false-positive target-unrelated peptides (TUPs) without any actual affinity towards the target remains a major problem of library screening. Selection-related TUPs may emerge because of binding to the components of the screening system rather than the target. Propagation-related TUPs may arise as a result of faster growth rate of some phage clones enabling them to outcompete slow-propagating clones. Amplification of the library between rounds of biopanning makes a significant contribution to the selection of phage clones with propagation advantage. Distinguishing nonspecific TUPs from true target binders is of particular importance for the translation of biopanning findings from basic research to clinical applications. Different experimental and in silico approaches are applied to assess the specificity of phage display-derived peptides towards the target. Bioinformatic tools are playing a rapidly growing role in the analysis of biopanning data and identification of target-irrelevant TUPs. Recent progress in the introduction of efficient strategies for TUP detection holds enormous promise for the discovery of clinically relevant cell- and tissue-homing peptides and paves the way for the development of novel targeted diagnostic and therapeutic platforms in pharmaceutical areas.

Synthetic Molecular Evolution of Membrane-Active Peptides

NASA Astrophysics Data System (ADS)

Wimley, William

The physical chemistry of membrane partitioning largely determines the function of membrane active peptides. Membrane-active peptides have potential utility in many areas, including in the cellular delivery of polar compounds, cancer therapy, biosensor design, and in antibacterial, antiviral and antifungal therapies. Yet, despite decades of research on thousands of known examples, useful sequence-structure-function relationships are essentially unknown. Because peptide-membrane interactions within the highly fluid bilayer are dynamic and heterogeneous, accounts of mechanism are necessarily vague and descriptive, and have little predictive power. This creates a significant roadblock to advances in the field. We are bypassing that roadblock with synthetic molecular evolution: iterative peptide library design and orthogonal high-throughput screening. We start with template sequences that have at least some useful activity, and create small, focused libraries using structural and biophysical principles to design the sequence space around the template. Orthogonal high-throughput screening is used to identify gain-of-function peptides by simultaneously selecting for several different properties (e.g. solubility, activity and toxicity). Multiple generations of iterative library design and screening have enabled the identification of membrane-active sequences with heretofore unknown properties, including clinically relevant, broad-spectrum activity against drug-resistant bacteria and enveloped viruses as well as pH-triggered macromolecular poration.

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya

Amino-acid mutations of Gly{sup 12} (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH{sub 2}) as a consensus sequence. KRpep-2 showedmore » more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. K{sub D} and IC{sub 50} values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH{sub 2}) that inhibited enzyme activity of K-Ras(G12D) with IC{sub 50} = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. - Highlights: • The first K-Ras(G12D)-selective inhibitory peptides were generated. • These peptides showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D) in compared to wild type K-Ras. • The peptide KRpep-2d suppressed downstream signal of K-Ras(G12D) and cell proliferations of cancer cell line A427.« less

The mzqLibrary – An open source Java library supporting the HUPO‐PSI quantitative proteomics standard

PubMed Central

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

2015-01-01

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/. PMID:26037908

Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

PubMed Central

Cha, S; Leung, P S; Van de Water, J; Tsuneyama, K; Joplin, R E; Ansari, A A; Nakanuma, Y; Schatz, P J; Cwirla, S; Fabris, L E; Neuberger, J M; Gershwin, M E; Coppel, R L

1996-01-01

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855289

A Combinatorial Platform for the Optimization of Peptidomimetic Methyl-Lysine Reader Antagonists

NASA Astrophysics Data System (ADS)

Barnash, Kimberly D.

Post-translational modification of histone N-terminal tails mediates chromatin compaction and, consequently, DNA replication, transcription, and repair. While numerous post-translational modifications decorate histone tails, lysine methylation is an abundant mark important for both gene activation and repression. Methyl-lysine (Kme) readers function through binding mono-, di-, or trimethyl-lysine. Chemical intervention of Kme readers faces numerous challenges due to the broad surface-groove interactions between readers and their cognate histone peptides; yet, the increasing interest in understanding chromatin-modifying complexes suggests tractable lead compounds for Kme readers are critical for elucidating the mechanisms of chromatin dysregulation in disease states and validating the druggability of these domains and complexes. The successful discovery of a peptide-derived chemical probe, UNC3866, for the Polycomb repressive complex 1 (PRC1) chromodomain Kme readers has proven the potential for selective peptidomimetic inhibition of reader function. Unfortunately, the systematic modification of peptides-to-peptidomimetics is a costly and inefficient strategy for target-class hit discovery against Kme readers. Through the exploration of biased chemical space via combinatorial on-bead libraries, we have developed two concurrent methodologies for Kme reader chemical probe discovery. We employ biased peptide combinatorial libraries as a hit discovery strategy with subsequent optimization via iterative targeted libraries. Peptide-to-peptidomimetic optimization through targeted library design was applied based on structure-guided library design around the interaction of the endogenous peptide ligand with three target Kme readers. Efforts targeting the WD40 reader EED led to the discovery of the 3-mer peptidomimetic ligand UNC5115 while combinatorial repurposing of UNC3866 for off-target chromodomains resulted in the discovery of UNC4991, a CDYL/2-selective ligand, and UNC4848, a MPP8 and CDYL/2 ligand. Ultimately, our efforts demonstrate the generalizability of a peptidomimetic combinatorial platform for the optimization of Kme reader ligands in a target class manner.

Denosumab mimics the natural decoy receptor osteoprotegerin by interacting with its major binding site on RANKL.

PubMed

Schieferdecker, Aneta; Voigt, Mareike; Riecken, Kristoffer; Braig, Friederike; Schinke, Thorsten; Loges, Sonja; Bokemeyer, Carsten; Fehse, Boris; Binder, Mascha

2014-08-30

Bone homeostasis critically relies on the RANKL-RANK-OPG axis which can be targeted by the fully human monoclonal antibody denosumab in conditions with increased bone resporption such as bone metastases. The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far. Here, we used random peptide phage display library screenings to identify the denosumab epitope on RANKL. Alignments of phage derived peptide sequences with RANKL suggested that this antibody recognized a linear epitope between position T233 and Y241. Mutational analysis confirmed the core residues as critical for this interaction. The spatial localization of this epitope on a 3-dimensional model of RANKL showed that it overlapped with the major binding sites of OPG and RANK on RANKL. We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.

Dynamic peptide libraries for the discovery of supramolecular nanomaterials

NASA Astrophysics Data System (ADS)

Pappas, Charalampos G.; Shafi, Ramim; Sasselli, Ivan R.; Siccardi, Henry; Wang, Tong; Narang, Vishal; Abzalimov, Rinat; Wijerathne, Nadeesha; Ulijn, Rein V.

2016-11-01

Sequence-specific polymers, such as oligonucleotides and peptides, can be used as building blocks for functional supramolecular nanomaterials. The design and selection of suitable self-assembling sequences is, however, challenging because of the vast combinatorial space available. Here we report a methodology that allows the peptide sequence space to be searched for self-assembling structures. In this approach, unprotected homo- and heterodipeptides (including aromatic, aliphatic, polar and charged amino acids) are subjected to continuous enzymatic condensation, hydrolysis and sequence exchange to create a dynamic combinatorial peptide library. The free-energy change associated with the assembly process itself gives rise to selective amplification of self-assembling candidates. By changing the environmental conditions during the selection process, different sequences and consequent nanoscale morphologies are selected.

Dynamic peptide libraries for the discovery of supramolecular nanomaterials.

PubMed

Pappas, Charalampos G; Shafi, Ramim; Sasselli, Ivan R; Siccardi, Henry; Wang, Tong; Narang, Vishal; Abzalimov, Rinat; Wijerathne, Nadeesha; Ulijn, Rein V

2016-11-01

Sequence-specific polymers, such as oligonucleotides and peptides, can be used as building blocks for functional supramolecular nanomaterials. The design and selection of suitable self-assembling sequences is, however, challenging because of the vast combinatorial space available. Here we report a methodology that allows the peptide sequence space to be searched for self-assembling structures. In this approach, unprotected homo- and heterodipeptides (including aromatic, aliphatic, polar and charged amino acids) are subjected to continuous enzymatic condensation, hydrolysis and sequence exchange to create a dynamic combinatorial peptide library. The free-energy change associated with the assembly process itself gives rise to selective amplification of self-assembling candidates. By changing the environmental conditions during the selection process, different sequences and consequent nanoscale morphologies are selected.

Clustering of disulfide-rich peptides provides scaffolds for hit discovery by phage display: application to interleukin-23.

PubMed

Barkan, David T; Cheng, Xiao-Li; Celino, Herodion; Tran, Tran T; Bhandari, Ashok; Craik, Charles S; Sali, Andrej; Smythe, Mark L

2016-11-23

Disulfide-rich peptides (DRPs) are found throughout nature. They are suitable scaffolds for drug development due to their small cores, whose disulfide bonds impart extraordinary chemical and biological stability. A challenge in developing a DRP therapeutic is to engineer binding to a specific target. This challenge can be overcome by (i) sampling the large sequence space of a given scaffold through a phage display library and by (ii) panning multiple libraries encoding structurally distinct scaffolds. Here, we implement a protocol for defining these diverse scaffolds, based on clustering structurally defined DRPs according to their conformational similarity. We developed and applied a hierarchical clustering protocol based on DRP structural similarity, followed by two post-processing steps, to classify 806 unique DRP structures into 81 clusters. The 20 most populated clusters comprised 85% of all DRPs. Representative scaffolds were selected from each of these clusters; the representatives were structurally distinct from one another, but similar to other DRPs in their respective clusters. To demonstrate the utility of the clusters, phage libraries were constructed for three of the representative scaffolds and panned against interleukin-23. One library produced a peptide that bound to this target with an IC 50 of 3.3 μM. Most DRP clusters contained members that were diverse in sequence, host organism, and interacting proteins, indicating that cluster members were functionally diverse despite having similar structure. Only 20 peptide scaffolds accounted for most of the natural DRP structural diversity, providing suitable starting points for seeding phage display experiments. Through selection of the scaffold surface to vary in phage display, libraries can be designed that present sequence diversity in architecturally distinct, biologically relevant combinations of secondary structures. We supported this hypothesis with a proof-of-concept experiment in which three phage libraries were constructed and panned against the IL-23 target, resulting in a single-digit μM hit and suggesting that a collection of libraries based on the full set of 20 scaffolds increases the potential to identify efficiently peptide binders to a protein target in a drug discovery program.

Toward a new and noninvasive diagnostic method of papillary thyroid cancer by using peptide vectorized contrast agents targeted to galectin-1.

PubMed

Fanfone, Deborah; Despretz, Nadège; Stanicki, Dimitri; Rubio-Magnieto, Jenifer; Fossépré, Mathieu; Surin, Mathieu; Rorive, Sandrine; Salmon, Isabelle; Vander Elst, Luce; Laurent, Sophie; Muller, Robert N; Saussez, Sven; Burtea, Carmen

2017-10-06

The incidence of papillary thyroid cancer has increased these last decades due to a better detection. High prevalence of nodules combined with the low incidence of thyroid cancers constitutes an important diagnostic challenge. We propose to develop an alternative diagnostic method to reduce the number of useless and painful thyroidectomies using a vectorized contrast agent for magnetic resonance imaging. Galectin-1 (gal-1), a protein overexpressed in well-differentiated thyroid cancer, has been targeted with a randomized linear 12-mer peptide library using the phage display technique. Selected peptides have been conjugated to ultrasmall superparamagnetic particles of iron oxide (USPIO). Peptides and their corresponding contrast agents have been tested in vitro for their specific binding and toxicity. Two peptides (P1 and P7) were selected according to their affinity toward gal-1. Their binding has been revealed by immunohistochemistry on human thyroid cancer biopsies, and they were co-localized with gal-1 by immunofluorescence on TPC-1 cell line. Both peptides induce a decrease in TPC-1 cells' adhesion to gal-1 immobilized on culture plates. After coupling to USPIO, the peptides preserved their affinity toward gal-1. Their specific binding has been corroborated by co-localization with gal-1 expressed by TPC-1 cells and by their ability to compete with anti-gal-1 antibody. The peptides and their USPIO derivatives produce no toxicity in HepaRG cells as determined by MTT assay. The vectorized contrast agents are potential imaging probes for thyroid cancer diagnosis. Moreover, the two gal-1-targeted peptides prevent cancer cell adhesion by interacting with the carbohydrate-recognition domain of gal-1.

PhD7Faster: predicting clones propagating faster from the Ph.D.-7 phage display peptide library.

PubMed

Ru, Beibei; 't Hoen, Peter A C; Nie, Fulei; Lin, Hao; Guo, Feng-Biao; Huang, Jian

2014-02-01

Phage display can rapidly discover peptides binding to any given target; thus, it has been widely used in basic and applied research. Each round of panning consists of two basic processes: Selection and amplification. However, recent studies have showed that the amplification step would decrease the diversity of phage display libraries due to different propagation capacity of phage clones. This may induce phages with growth advantage rather than specific affinity to appear in the final experimental results. The peptides displayed by such phages are termed as propagation-related target-unrelated peptides (PrTUPs). They would mislead further analysis and research if not removed. In this paper, we describe PhD7Faster, an ensemble predictor based on support vector machine (SVM) for predicting clones with growth advantage from the Ph.D.-7 phage display peptide library. By using reduced dipeptide composition (ReDPC) as features, an accuracy (Acc) of 79.67% and a Matthews correlation coefficient (MCC) of 0.595 were achieved in 5-fold cross-validation. In addition, the SVM-based model was demonstrated to perform better than several representative machine learning algorithms. We anticipate that PhD7Faster can assist biologists to exclude potential PrTUPs and accelerate the finding of specific binders from the popular Ph.D.-7 library. The web server of PhD7Faster can be freely accessed at http://immunet.cn/sarotup/cgi-bin/PhD7Faster.pl.

Enhanced secretion of natto phytase by Bacillus subtilis.

PubMed

Tsuji, Shogo; Tanaka, Kosei; Takenaka, Shinji; Yoshida, Ken-ichi

2015-01-01

Phytases comprise a group of phosphatases that trim inorganic phosphates from phytic acid (IP6). In this study, we aimed to achieve the efficient secretion of phytase by Bacillus subtilis. B. subtilis laboratory standard strain 168 and its derivatives exhibit no phytase activity, whereas a natto starter secretes phytase actively. The natto phytase gene was cloned into strain RIK1285, a protease-defective derivative of 168, to construct a random library of its N-terminal fusions with 173 different signal peptides (SPs) identified in the 168 genome. The library was screened to assess the efficiency of phytase secretion based on clear zones around colonies on plates, which appeared when IP6 was hydrolyzed. The pbp SP enhanced the secretion of the natto phytase most efficiently, i.e. twice that of the original SP. Thus, the secreted natto phytase was purified and found to remove up to 3 phosphates from IP6.

Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line

PubMed Central

Stiefelhagen, Marius; Sellner, Leopold; Kleinschmidt, Jürgen A; Jauch, Anna; Laufs, Stephanie; Wenz, Frederik; Zeller, W Jens; Fruehauf, Stefan; Veldwijk, Marlon R

2008-01-01

Background For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants. Methods To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls. Results Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% ± 2% GFP+ cells; Lama84: 36-fold, 29% ± 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed. Conclusion Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors. PMID:18789140

Optimization and high-throughput screening of antimicrobial peptides.

PubMed

Blondelle, Sylvie E; Lohner, Karl

2010-01-01

While a well-established process for lead compound discovery in for-profit companies, high-throughput screening is becoming more popular in basic and applied research settings in academia. The development of combinatorial libraries combined with easy and less expensive access to new technologies have greatly contributed to the implementation of high-throughput screening in academic laboratories. While such techniques were earlier applied to simple assays involving single targets or based on binding affinity, they have now been extended to more complex systems such as whole cell-based assays. In particular, the urgent need for new antimicrobial compounds that would overcome the rapid rise of drug-resistant microorganisms, where multiple target assays or cell-based assays are often required, has forced scientists to focus onto high-throughput technologies. Based on their existence in natural host defense systems and their different mode of action relative to commercial antibiotics, antimicrobial peptides represent a new hope in discovering novel antibiotics against multi-resistant bacteria. The ease of generating peptide libraries in different formats has allowed a rapid adaptation of high-throughput assays to the search for novel antimicrobial peptides. Similarly, the availability nowadays of high-quantity and high-quality antimicrobial peptide data has permitted the development of predictive algorithms to facilitate the optimization process. This review summarizes the various library formats that lead to de novo antimicrobial peptide sequences as well as the latest structural knowledge and optimization processes aimed at improving the peptides selectivity.

Differential gene expression profiles in the venom gland/sac of Eumenes pomiformis (Hymenoptera: Eumenidae).

PubMed

Baek, Ji Hyeong; Lee, Si Hyeock

2010-06-01

To search for novel transcripts encoding biologically active venom components, a subtractive cDNA library specific to the venom gland and sac (gland/sac) of a solitary hunting wasp species, Eumenes pomiformis Fabricius (1781), was constructed by suppression subtractive hybridization. A total of 541 expressed sequence tags (ESTs) were clustered and assembled into 102 contigs (31 multiple sequences and 71 singletons). In total, 37 cDNAs were found in the library via BLASTx searching and manual annotation. Eight contigs (337 ESTs) encoding short venom peptides (10 to 16 amino acids) occupied 62% of the library. The deduced amino acid sequence (78 amino acids) of a novel venom peptide transcript shared sequence similarity with trypsin inhibitors and dendrotoxin-like venom peptides known to be K(+) channel blockers, implying that this novel peptide may play a role in the paralysis of prey. In addition to phospholipase A2 and hyaluronidase, which are known to be the main components of wasp venoms, several transcripts encoding enzymes, including three metallopeptidases and a decarboxylase likely involved in the processing and activation of venomous proteins, peptides, amines, and neurotransmitters, were also isolated from the library. The presence of a transcript encoding a putative insulin/insulin-like peptide binding protein suggests that solitary hunting wasps use their venom to control their prey, leading to larval growth cessation. The abundance of these venom components in the venom gland/sac and in the alimentary canal was confirmed by quantitative real-time PCR. Discovery of venom gland/sac-specific transcripts should promote further studies on biologically active components in the venom of solitary hunting wasps. Copyright 2010 Elsevier Ltd. All rights reserved.

Discovering Peptide Inhibitors of Human Squalene Synthase Through Screening the Phage-Displayed Cyclic Peptide c7c Library.

PubMed

Shiuan, David; Chen, Yue-Hao; Lin, Hwan-Kang; Huang, Kao-Jean; Tai, Da-Fu; Chang, Ding-Kwo

2016-06-01

Many drugs for the treatment of hypercholesterolemia are targeting the enzymes involved in human cholesterol biosynthesis pathway. Squalene synthase, the rate-limiting enzyme located at the downstream of cholesterol synthesis pathway, has become a better candidate to develop next-generation hypocholesterolemia drugs. In the present study, we cloned and expressed the recombinant human squalene synthase (hSQS) as the lure to isolate potential peptide inhibitors from screening the conformation-constrained phage-displayed cyclic peptide c7c library. Their binding capabilities were further estimated by ELISA. Their pharmaceutical potentials were then analyzed through molecular modeling and the ADMET property evaluations. Four ennea-peptides and nine tetra-peptides were finally synthesized to evaluate their inhibitory potentials toward hSQS. The results indicate that the ennea-peptide CLSPHSMFC, tetra-peptides SMFC, CKTE, and WHQW can effectively inhibit hSQS activities (IC50 values equal to 64, 76, 87, and 90 μM, respectively). These peptides may have potentials to develop future cholesterol-lowering therapeutics. The ligand-protein interaction analysis also reveals that the inner hydrophobic pocket could be a more critical site of hSQS.

Sample preparation for sequencing hits from one-bead-one-peptide combinatorial libraries by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Martínez-Ceron, María C; Giudicessi, Silvana L; Marani, Mariela M; Albericio, Fernando; Cascone, Osvaldo; Erra-Balsells, Rosa; Camperi, Silvia A

2010-05-15

Optimization of bead analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after the screening of one-bead-one-peptide combinatorial libraries was achieved, involving the fine-tuning of the whole process. Guanidine was replaced by acetonitrile (MeCN)/acetic acid (AcOH)/water (H(2)O), improving matrix crystallization. Peptide-bead cleavage with NH(4)OH was cheaper and safer than, yet as efficient as, NH(3)/tetrahydrofuran (THF). Peptide elution in microtubes instead of placing the beads in the sample plate yielded more sample aliquots. Successive dry layers deposit sample preparation was better than the dried droplet method. Among the matrices analyzed, alpha-cyano-4-hydroxycinnamic acid resulted in the best peptide ion yield. Cluster formation was minimized by the addition of additives to the matrix. Copyright 2010 Elsevier Inc. All rights reserved.

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands.

PubMed

Das, Samir; Nag, Arundhati; Liang, JingXin; Bunck, David N; Umeda, Aiko; Farrow, Blake; Coppock, Matthew B; Sarkes, Deborah A; Finch, Amethist S; Agnew, Heather D; Pitram, Suresh; Lai, Bert; Yu, Mary Beth; Museth, A Katrine; Deyle, Kaycie M; Lepe, Bianca; Rodriguez-Rivera, Frances P; McCarthy, Amy; Alvarez-Villalonga, Belen; Chen, Ann; Heath, John; Stratis-Cullum, Dimitra N; Heath, James R

2015-11-02

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transformation of Human Cathelicidin LL-37 into Selective, Stable, and Potent Antimicrobial Compounds

PubMed Central

2015-01-01

This Letter reports a family of novel antimicrobial compounds obtained by combining peptide library screening with structure-based design. Library screening led to the identification of a human LL-37 peptide resistant to chymotrypsin. This d-amino-acid-containing peptide template was active against Escherichia coli but not methicillin-resistant Staphylococcus aureus (MRSA). It possesses a unique nonclassic amphipathic structure with hydrophobic defects. By repairing the hydrophobic defects, the peptide (17BIPHE2) gained activity against the ESKAPE pathogens, including Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species. In vitro, 17BIPHE2 could disrupt bacterial membranes and bind to DNA. In vivo, the peptide prevented staphylococcal biofilm formation in a mouse model of catheter-associated infection. Meanwhile, it boosted the innate immune response to further combat the infection. Because these peptides are potent, cell-selective, and stable to several proteases, they may be utilized to combat one or more ESKAPE pathogens. PMID:25061850

Sensitive electrospray mass spectrometry analysis of one-bead-one-compound peptide libraries labeled by quaternary ammonium salts.

PubMed

Bąchor, Remigiusz; Cydzik, Marzena; Rudowska, Magdalena; Kluczyk, Alicja; Stefanowicz, Piotr; Szewczuk, Zbigniew

2012-08-01

A rapid and straightforward method for high-throughput analysis of single resin beads from one-bead-one-compound combinatorial libraries with high resolution electrospray ionization tandem mass spectrometry (HR ESI-MS/MS) is presented. The application of an efficient method of peptide derivatization by quaternary ammonium salts (QAS) formation increases ionization efficiency and reduces the detection limit, allowing analysis of trace amounts of compounds by ESI-MS. Peptides, synthesized on solid support, contain a new cleavable linker composed of a Peg spacer (9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid), lysine with ɛ-amino group marked by the N,N,N-triethylglycine salt, and methionine, which makes possible the selective cleavage by cyanogen bromide. Even a small portion of peptides derivatized by QAS cleaved from a single resin bead is sufficient for sequencing by HR ESI-MS/MS experiments. The developed strategy was applied to a small training library of α chymotrypsin substrates. The obtained results confirm the applicability of the proposed method in combinatorial chemistry.

Entomopathogenic bacteria use multiple mechanisms for bioactive peptide library design

NASA Astrophysics Data System (ADS)

Cai, Xiaofeng; Nowak, Sarah; Wesche, Frank; Bischoff, Iris; Kaiser, Marcel; Fürst, Robert; Bode, Helge. B.

2017-04-01

The production of natural product compound libraries has been observed in nature for different organisms such as bacteria, fungi and plants; however, little is known about the mechanisms generating such chemically diverse libraries. Here we report mechanisms leading to the biosynthesis of the chemically diverse rhabdopeptide/xenortide peptides (RXPs). They are exclusively present in entomopathogenic bacteria of the genera Photorhabdus and Xenorhabdus that live in symbiosis with nematodes delivering them to insect prey, which is killed and utilized for nutrition by both nematodes and bacteria. Chemical diversity of the biologically active RXPs results from a combination of iterative and flexible use of monomodular nonribosomal peptide synthetases including substrate promiscuity, enzyme cross-talk and enzyme stoichiometry as shown by in vivo and in vitro experiments. Together, this highlights several of nature's methods for diversification, or evolution, of natural products and sheds light on the biosynthesis of the bioactive RXPs.

Anti-dengue virus serotype 2 activity and mode of action of a novel peptide.

PubMed

Chew, M-F; Tham, H-W; Rajik, M; Sharifah, S H

2015-10-01

To identify a novel antiviral peptide against dengue virus serotype 2 (DENV-2) by screening a phage display peptide library and to evaluate its in vitro antiviral activity and mode of action. A phage display peptide library was biopanned against purified DENV-2 and resulted in the identification and selection of a peptide (peptide gg-ww) for further investigation. ELISA was performed, and peptide gg-ww was shown to possess the highest binding affinity against DENV-2. Thus, peptide gg-ww was synthesized for cytotoxicity and antiviral assays. Virus plaque reduction assay, real-time PCR and immunofluorescence assay were used to investigate the inhibitory effect of peptide gg-ww on DENV-2 infection in Vero cells. Three different assays (pre-, simultaneous and post-treatments assays) were performed to investigate the peptide's mode of action. Results indicated that peptide gg-ww possessed strong antiviral activity with a ~96% inhibition rate, which was achieved at 250 μmol l(-1) . Viral replication was inhibited during a simultaneous treatment assay, indicating that the entry of the virus was impeded by this peptide. Peptide gg-ww displayed antiviral action against DENV-2 by targeting an early stage of viral replication (i.e. during viral entry). Peptide gg-ww may represent a new therapeutic candidate for the treatment of DENV infections and is a potential candidate to be developed as a peptide drug. © 2015 The Society for Applied Microbiology.

Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge

PubMed Central

Kogot, Joshua M.; Zhang, Yanting; Moore, Stephen J.; Pagano, Paul; Stratis-Cullum, Dimitra N.; Chang-Yen, David; Turewicz, Marek; Pellegrino, Paul M.; de Fusco, Andre; Soh, H. Tom; Stagliano, Nancy E.

2011-01-01

Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×1010 members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS. PMID:22140433

Identification of polypeptides with selective affinity to intact mouse cerebellar granule neurons from a random peptide-presenting phage library.

PubMed

Hou, Sheng T; Dove, Mike; Anderson, Erica; Zhang, Jiangbing; MacKenzie, C Roger

2004-09-30

Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.

Selection of a platinum-binding sequence in a loop of a four-helix bundle protein.

PubMed

Yagi, Sota; Akanuma, Satoshi; Kaji, Asumi; Niiro, Hiroya; Akiyama, Hayato; Uchida, Tatsuya; Yamagishi, Akihiko

2018-02-01

Protein-metal hybrids are functional materials with various industrial applications. For example, a redox enzyme immobilized on a platinum electrode is a key component of some biofuel cells and biosensors. To create these hybrid materials, protein molecules are bound to metal surfaces. Here, we report the selection of a novel platinum-binding sequence in a loop of a four-helix bundle protein, the Lac repressor four-helix protein (LARFH), an artificial protein in which four identical α-helices are connected via three identical loops. We created a genetic library in which the Ser-Gly-Gln-Gly-Gly-Ser sequence within the first inter-helical loop of LARFH was semi-randomly mutated. The library was then subjected to selection for platinum-binding affinity by using the T7 phage display method. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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. Copyright © 2015 Elsevier B.V. All rights reserved.

One-Bead-Two-Compound Thioether Bridged Macrocyclic γ-AApeptide Screening Library against EphA2.

PubMed

Shi, Yan; Challa, Sridevi; Sang, Peng; She, Fengyu; Li, Chunpu; Gray, Geoffrey M; Nimmagadda, Alekhya; Teng, Peng; Odom, Timothy; Wang, Yan; van der Vaart, Arjan; Li, Qi; Cai, Jianfeng

2017-11-22

Identification of molecular ligands that recognize peptides or proteins is significant but poses a fundamental challenge in chemical biology and biomedical sciences. Development of cyclic peptidomimetic library is scarce, and thus discovery of cyclic peptidomimetic ligands for protein targets is rare. Herein we report the unprecedented one-bead-two-compound (OBTC) combinatorial library based on a novel class of the macrocyclic peptidomimetics γ-AApeptides. In the library, we utilized the coding peptide tags synthesized with Dde-protected α-amino acids, which were orthogonal to solid phase synthesis of γ-AApeptides. Employing the thioether linkage, the desired macrocyclic γ-AApeptides were found to be effective for ligand identification. Screening the library against the receptor tyrosine kinase EphA2 led to the discovery of one lead compound that tightly bound to EphA2 (K d = 81 nM) and potently antagonized EphA2-mediated signaling. This new approach of macrocyclic peptidomimetic library may lead to a novel platform for biomacromolecular surface recognition and function modulation.

Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

PubMed

Wang, Hong; Brautigan, David L

2006-11-01

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library.

PubMed

Buzatti, Andréia; Fernandez, Arnielis Diaz; Arenal, Amilcar; Pereira, Erlán; Monteiro, Alda Lucia Gomes; Molento, Marcelo Beltrão

2018-05-24

The aim of this study was to evaluate phage display technology for mapping Haemonchus contortus mimotopes. We screened the PhD-7 Phage Display Peptide Library Kit with a sheep polyclonal antibody against H. contortus. After four rounds of selection, 50 phage peptide clones were selected by biopanning and sequenced. Two clones displaying peptide mimotopes of H. contortus proteins were chosen for sheep immunization: clone 6 - mimotope of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and clone 17 - mimotope of a disorganized muscle family member (Dim 1). Twelve sheep were allocated into 3 groups of 4 animals as follow: G1: control group; G2/GAPDH: immunized with clone 6; and G3/Dim1: immunized with clone 17. Four immunizations were performed at intervals of seven days (0, 7, 14, and 21 days). On day 28 post initial vaccination, all groups were orally challenged with 2500 H. contortus infective larvae. The mimotope peptides selected by phage display were recognized by IgG from sheep naturaly infected with H. contortus. The immunization protocol showed an increasein IgG anti-M13 phage titers, but no effect was observed in IgG-specific for the anti-mimotope peptides. This is the first report of successful use of a phage display library for the identification of mimotopes of H. contortus proteins.

Corruption of phage-display libraries by target-unrelated clones: Diagnosis and countermeasures

PubMed Central

Thomas, William D.; Golomb, Miriam; Smith, George P.

2010-01-01

Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior, and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phage (TUPs), that lack the target behavior. Many TUPs are propagation-related; they have mutations conferring a growth advantage, and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. PMID:20692225

Control of Surface Chemistry, Substrate Stiffness, and Cell Function in a Novel Terpolymer Methacrylate Library

PubMed Central

Joy, Abraham; Cohen, Daniel M.; Luk, Arnold; Anim-Danso, Emmanuel; Chen, Christopher; Kohn, Joachim

2011-01-01

A focused library of methacrylate terpolymers was synthesized to explore the effects of varying surface chemistry and adhesive peptide ligands on cell function. The chemical diversity of methacrylate monomers enabled construction of a library of polymers in which one can systematically vary the chemical composition to achieve a wide range of contact angle, Young's modulus, and Tg values. Furthermore, the materials were designed to allow surface immobilization of bioactive peptides. We then examined the effects of these material compositions on protein adsorption and cell attachment, proliferation, and differentiation. We observed that chemical composition of the polymers was an important determinant for NIH 3T3 cell attachment and proliferation, as well as human mesenchymal stem cell differentiation, and correlated directly with the ability of the polymers to adsorb proteins that mediate cell adhesion. Importantly, functionalization of the methacrylate terpolymer library with an adhesive GRGDS peptide normalized cellular responses. RGD-functionalized polymers uniformly exhibited robust attachment, proliferation, and differentiation irrespective of the underlying substrate chemistry. These studies provide a library-based approach to rapidly explore the biological functionality of biomaterials with a wide range of compositions, and highlights the importance of cell and protein cell adhesion in predicting their performance. PMID:21226505

Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing.

PubMed

Liu, Gary W; Livesay, Brynn R; Kacherovsky, Nataly A; Cieslewicz, Maryelise; Lutz, Emi; Waalkes, Adam; Jensen, Michael C; Salipante, Stephen J; Pun, Suzie H

2015-08-19

Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying "parasitic sequences" and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively activated M2 macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences.

An integrated vector system for cellular studies of phage display-derived peptides.

PubMed

Voss, Stephan D; DeGrand, Alec M; Romeo, Giulio R; Cantley, Lewis C; Frangioni, John V

2002-09-15

Peptide phage display is a method by which large numbers of diverse peptides can be screened for binding to a target of interest. Even when successful, the rate-limiting step is usually validation of peptide bioactivity using living cells. In this paper, we describe an integrated system of vectors that expedites both the screening and the characterization processes. Library construction and screening is performed using an optimized type 3 phage display vector, mJ(1), which is shown to accept peptide libraries of at least 23 amino acids in length. Peptide coding sequences are shuttled from mJ(1) into one of three families of mammalian expression vectors for cell physiological studies. The vector pAL(1) expresses phage display-derived peptides as Gal4 DNA binding domain fusion proteins for transcriptional activation studies. The vectors pG(1), pG(1)N, and pG(1)C express phage display-derived peptides as green fluorescent protein fusions targeted to the entire cell, nucleus, or cytoplasm, respectively. The vector pAP(1) expresses phage display-derived peptides as fusions to secreted placental alkaline phosphatase. Such enzyme fusions can be used as highly sensitive affinity reagents for high-throughput assays and for cloning of peptide-binding cell surface receptors. Taken together, this system of vectors should facilitate the development of phage display-derived peptides into useful biomolecules.

Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

PubMed

Seo, Moon-Hyeong; Nim, Satra; Jeon, Jouhyun; Kim, Philip M

2017-01-01

Protein-protein interactions are essential to cellular functions and signaling pathways. We recently combined bioinformatics and custom oligonucleotide arrays to construct custom-made peptide-phage libraries for screening peptide-protein interactions, an approach we call proteomic peptide-phage display (ProP-PD). In this chapter, we describe protocols for phage display for the identification of natural peptide binders for a given protein. We finally describe deep sequencing for the analysis of the proteomic peptide-phage display.

A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities.

PubMed

Hitomi, Kiyotaka; Kitamura, Miyako; Alea, Mileidys Perez; Ceylan, Ismail; Thomas, Vincent; El Alaoui, Saïd

2009-11-15

Transglutaminase (TGase) is an enzyme that catalyzes both isopeptide cross-linking and incorporation of primary amines into proteins. Eight TGases have been identified in humans, and each of these TGases has a unique tissue distribution and physiological significance. Although several assays for TGase enzymatic activity have been reported, it has been difficult to establish an assay for discriminating each of these different TGase activities. Using a random peptide library, we recently identified the preferred substrate sequences for three major TGases: TGase 1, TGase 2, and factor XIII. In this study, we use these substrates in specific tests for measuring the activities of TGase 1 and factor XIII.

Sequence homology between HLA-bound cytomegalovirus and human peptides: A potential trigger for alloreactivity

PubMed Central

Koparde, Vishal N.; Jameson-Lee, Maximilian; Elnasseh, Abdelrhman G.; Scalora, Allison F.; Kobulnicky, David J.; Serrano, Myrna G.; Roberts, Catherine H.; Buck, Gregory A.; Neale, Michael C.; Nixon, Daniel E.; Toor, Amir A.

2017-01-01

Human cytomegalovirus (hCMV) reactivation may often coincide with the development of graft-versus-host-disease (GVHD) in stem cell transplantation (SCT). Seventy seven SCT donor-recipient pairs (DRP) (HLA matched unrelated donor (MUD), n = 50; matched related donor (MRD), n = 27) underwent whole exome sequencing to identify single nucleotide polymorphisms (SNPs) generating alloreactive peptide libraries for each DRP (9-mer peptide-HLA complexes); Human CMV CROSS (Cross-Reactive Open Source Sequence) database was compiled from NCBI; HLA class I binding affinity for each DRPs HLA was calculated by NetMHCpan 2.8 and hCMV- derived 9-mers algorithmically compared to the alloreactive peptide-HLA complex libraries. Short consecutive (≥6) amino acid (AA) sequence homology matching hCMV to recipient peptides was considered for HLA-bound-peptide (IC50<500nM) cross reactivity. Of the 70,686 hCMV 9-mers contained within the hCMV CROSS database, an average of 29,658 matched the MRD DRP alloreactive peptides and 52,910 matched MUD DRP peptides (p<0.001). In silico analysis revealed multiple high affinity, immunogenic CMV-Human peptide matches (IC50<500 nM) expressed in GVHD-affected tissue-specific manner. hCMV+GVHD was found in 18 patients, 13 developing hCMV viremia before GVHD onset. Analysis of patients with GVHD identified potential cross reactive peptide expression within affected organs. We propose that hCMV peptide sequence homology with human alloreactive peptides may contribute to the pathophysiology of GVHD. PMID:28800601

Sequence homology between HLA-bound cytomegalovirus and human peptides: A potential trigger for alloreactivity.

PubMed

Hall, Charles E; Koparde, Vishal N; Jameson-Lee, Maximilian; Elnasseh, Abdelrhman G; Scalora, Allison F; Kobulnicky, David J; Serrano, Myrna G; Roberts, Catherine H; Buck, Gregory A; Neale, Michael C; Nixon, Daniel E; Toor, Amir A

2017-01-01

Human cytomegalovirus (hCMV) reactivation may often coincide with the development of graft-versus-host-disease (GVHD) in stem cell transplantation (SCT). Seventy seven SCT donor-recipient pairs (DRP) (HLA matched unrelated donor (MUD), n = 50; matched related donor (MRD), n = 27) underwent whole exome sequencing to identify single nucleotide polymorphisms (SNPs) generating alloreactive peptide libraries for each DRP (9-mer peptide-HLA complexes); Human CMV CROSS (Cross-Reactive Open Source Sequence) database was compiled from NCBI; HLA class I binding affinity for each DRPs HLA was calculated by NetMHCpan 2.8 and hCMV- derived 9-mers algorithmically compared to the alloreactive peptide-HLA complex libraries. Short consecutive (≥6) amino acid (AA) sequence homology matching hCMV to recipient peptides was considered for HLA-bound-peptide (IC50<500nM) cross reactivity. Of the 70,686 hCMV 9-mers contained within the hCMV CROSS database, an average of 29,658 matched the MRD DRP alloreactive peptides and 52,910 matched MUD DRP peptides (p<0.001). In silico analysis revealed multiple high affinity, immunogenic CMV-Human peptide matches (IC50<500 nM) expressed in GVHD-affected tissue-specific manner. hCMV+GVHD was found in 18 patients, 13 developing hCMV viremia before GVHD onset. Analysis of patients with GVHD identified potential cross reactive peptide expression within affected organs. We propose that hCMV peptide sequence homology with human alloreactive peptides may contribute to the pathophysiology of GVHD.

Detection of Listeria monocytogenes with short peptide fragments from class IIa bacteriocins as recognition elements.

PubMed

Azmi, Sarfuddin; Jiang, Keren; Stiles, Michael; Thundat, Thomas; Kaur, Kamaljit

2015-03-09

We employed a direct peptide-bacteria binding assay to screen peptide fragments for high and specific binding to Listeria monocytogenes. Peptides were screened from a peptide array library synthesized on cellulose membrane. Twenty four peptide fragments (each a 14-mer) were derived from three potent anti-listerial peptides, Leucocin A, Pediocin PA1, and Curvacin A, that belong to class IIa bacteriocins. Fragment Leu10 (GEAFSAGVHRLANG), derived from the C-terminal region of Leucocin A, displayed the highest binding among all of the library fragments toward several pathogenic Gram-positive bacteria, including L. monocytogenes, Enterococcus faecalis, and Staphylococcus aureus. The specific binding of Leu10 to L. monocytogenes was further validated using microcantilever (MCL) experiments. Microcantilevers coated with gold were functionalized with peptides by chemical conjugation using a cysteamine linker to yield a peptide density of ∼4.8×10(-3) μmol/cm2 for different peptide fragments. Leu10 (14-mer) functionalized MCL was able to detect Listeria with same sensitivity as that of Leucocin A (37-mer) functionalized MCL, validating the use of short peptide fragments in bacterial detection platforms. Fragment Leu10 folded into a helical conformation in solution, like that of native Leucocin A, suggesting that both Leu10 and Leucocin A may employ a similar mechanism for binding target bacteria. The results show that peptide-conjugated microcantilevers can function as highly sensitive platforms for Listeria detection and hold potential to be developed as biosensors for pathogenic bacteria.

Non-natural amino acid peptide microarrays to discover Ebola virus glycoprotein ligands.

PubMed

Rabinowitz, Joshua A; Lainson, John C; Johnston, Stephen Albert; Diehnelt, Chris W

2018-02-06

We demonstrate a platform to screen a virus pseudotyped with Ebola virus glycoprotein (GP) against a library of peptides that contain non-natural amino acids to develop GP affinity ligands. This system could be used for rapid development of peptide-based antivirals for other emerging or neglected tropical infectious diseases.

The protein and peptide mediated syntheses of non-biologically-produced oxide materials

NASA Astrophysics Data System (ADS)

Dickerson, Matthew B.

Numerous examples exist in nature of organisms which have evolved the ability to produce sophisticated structures composed of inorganic minerals. Studies of such biomineralizing organisms have suggested that specialized biomolecules are, in part, responsible for the controlled formation of these structures. The research detailed in this dissertation is focused on the use of biomolecules (i.e., peptides and proteins) to form non-biologically produced materials under mild reaction conditions (i.e., neutral pH, aqueous solutions, and room temperature). The peptides utilized in the studies detailed in this dissertation were identified through the screening of single crystal rutile TiO2 substrates or Ge powder with a phagedisplayed peptide library. Twenty-one peptides were identified which possessed an affinity for Ge. Three of these twenty one peptides were tested for germania precipitation activity. Those peptides possessing a basic isoelectric point as well as hydroxyl- and imidazole-containing amino acid residues were found to be the most effective in precipitating amorphous germania from an alkoxide precursor. The phage-displayed peptide library screening of TiO2 substrates yielded twenty peptides. Four of these peptides, which were heavily enriched in histidine and/or basic amino acid residues, were found to possess signficant titania precipitation activity. The activity of these peptides was found to correlate with the number of positive charges they carried. The sequence of the most active of the library-identified peptides was modified to yield two additional peptides. The titania precipitation activity of these designed peptides was higher than the parent peptide, with reduced pH dependence. The titania materials generated by the library-identified and designed peptides were found to be composed of amorphous titania as well as <10 nm anatase and/or monoclinic TiO2 crystallites. The production of titania and zirconia resulting from the interaction of the cationic enzyme, hen egg white lysozyme, with Ti- or Zr-lactate precursors is also presented in this dissertation. Lysozyme was found to entrap itself in an active form within the nanoparticles of amorphous titania or zirconia precipitated by this protein under ambient conditions. The lysozyme synthesized titania was observed to be superior to the lysozyme-zirconia materials in preserving the activity of the enzyme under denaturing conditions. Four recombinant proteins, derived from the amino acid sequences of proteins (silaffins) associated with biosilicification in diatoms, were also investigated for titania precipitation activity. The two most basic of these recombinant silaffins, rSil1L and rSilC, were able to induce the formation of titania. The titania precipitates generated by rSil1L were found to be similar to those produced by the phage-displayed library identified peptides. The second recombinant silaffin, rSilC, was found to produce hollow spheres of titania, which, following dehydration, were observed to transform into larger, solid spheres composed of radially aligned columns of rutile TiO2. The highly repetitive nature of the rSilC's amino acid sequence is believed to be responsible for the differences in TiO2 polymorph generated by the different recombinant silaffins and peptides. This dissertation also details research conducted on the formation of titania utilizing rSilC conjugated to synthetic and biogenic silica surfaces. These silica surfaces were functionalized with a newly developed drendritic growth technique. The dendritic functional-group amplification process was demonstrated to increase the loading of hexahisitidine tagged proteins on silica surfaces by more than 40%, as compared to traditional immobilization procedures. The higher loadings of rSilC provided by this dendritic growth method were observed to have a positive impact on the extent of surface mineralization. The titania formed by immobilized rSilC was observed to be composed of amorphous and crystalline TiO2.

Analysing the substrate multispecificity of a proton-coupled oligopeptide transporter using a dipeptide library

PubMed Central

Ito, Keisuke; Hikida, Aya; Kawai, Shun; Lan, Vu Thi Tuyet; Motoyama, Takayasu; Kitagawa, Sayuri; Yoshikawa, Yuko; Kato, Ryuji; Kawarasaki, Yasuaki

2013-01-01

Peptide uptake systems that involve members of the proton-coupled oligopeptide transporter (POT) family are conserved across all organisms. POT proteins have characteristic substrate multispecificity, with which one transporter can recognize as many as 8,400 types of di/tripeptides and certain peptide-like drugs. Here we characterize the substrate multispecificity of Ptr2p, a major peptide transporter of Saccharomyces cerevisiae, using a dipeptide library. The affinities (Ki) of di/tripeptides toward Ptr2p show a wide distribution range from 48 mM to 0.020 mM. This substrate multispecificity indicates that POT family members have an important role in the preferential uptake of vital amino acids. In addition, we successfully establish high performance ligand affinity prediction models (97% accuracy) using our comprehensive dipeptide screening data in conjunction with simple property indices for describing ligand molecules. Our results provide an important clue to the development of highly absorbable peptides and their derivatives including peptide-like drugs. PMID:24060756

Enhanced Prediction of Src Homology 2 (SH2) Domain Binding Potentials Using a Fluorescence Polarization-derived c-Met, c-Kit, ErbB, and Androgen Receptor Interactome*

PubMed Central

Leung, Kin K.; Hause, Ronald J.; Barkinge, John L.; Ciaccio, Mark F.; Chuu, Chih-Pin; Jones, Richard B.

2014-01-01

Many human diseases are associated with aberrant regulation of phosphoprotein signaling networks. Src homology 2 (SH2) domains represent the major class of protein domains in metazoans that interact with proteins phosphorylated on the amino acid residue tyrosine. Although current SH2 domain prediction algorithms perform well at predicting the sequences of phosphorylated peptides that are likely to result in the highest possible interaction affinity in the context of random peptide library screens, these algorithms do poorly at predicting the interaction potential of SH2 domains with physiologically derived protein sequences. We employed a high throughput interaction assay system to empirically determine the affinity between 93 human SH2 domains and phosphopeptides abstracted from several receptor tyrosine kinases and signaling proteins. The resulting interaction experiments revealed over 1000 novel peptide-protein interactions and provided a glimpse into the common and specific interaction potentials of c-Met, c-Kit, GAB1, and the human androgen receptor. We used these data to build a permutation-based logistic regression classifier that performed considerably better than existing algorithms for predicting the interaction potential of several SH2 domains. PMID:24728074

Biologically-Inspired Peptide Reagents for Enhancing IMS-MS Analysis of Carbohydrates

NASA Astrophysics Data System (ADS)

Bohrer, Brian C.; Clemmer, David E.

2011-09-01

The binding properties of a peptidoglycan recognition protein are translated via combinatorial chemistry into short peptides. Non-adjacent histidine, tyrosine, and arginine residues in the protein's binding cleft that associate specifically with the glycan moiety of a peptidoglycan substrate are incorporated into linear sequences creating a library of 27 candidate tripeptide reagents (three possible residues permutated across three positions). Upon electrospraying the peptide library and carbohydrate mixtures, some noncovalent complexes are observed. The binding efficiencies of the peptides vary according to their amino acid composition as well as the disaccharide linkage and carbohydrate ring-type. In addition to providing a charge-carrier for the carbohydrate, peptide reagents can also be used to differentiate carbohydrate isomers by ion mobility spectrometry. The utility of these peptide reagents as a means of enhancing ion mobility analysis of carbohydrates is illustrated by examining four glucose-containing disaccharide isomers, including a pair that is not resolved by ion mobility alone. The specificity and stoichiometry of the peptide-carbohydrate complexes are also investigated. Trihistidine demonstrates both suitable binding efficiency and successful resolution of disaccharides isomers, suggesting it may be a useful reagent in IMS analyses of carbohydrates.

Specificity of Mimotope-Induced Anti-High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) Antibodies Does Not Ensure Biological Activity

PubMed Central

Hofstetter, Gerlinde; Balazs, Nina; Smole, Ursula; Ferrone, Soldano; Scheiner, Otto; Breiteneder, Heimo; Pehamberger, Hubert; Wagner, Stefan

2011-01-01

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes. PMID:21573118

Specificity of mimotope-induced anti-high molecular weight-melanoma associated antigen (HMW-MAA) antibodies does not ensure biological activity.

PubMed

Latzka, Julia; Gaier, Sonja; Hofstetter, Gerlinde; Balazs, Nina; Smole, Ursula; Ferrone, Soldano; Scheiner, Otto; Breiteneder, Heimo; Pehamberger, Hubert; Wagner, Stefan

2011-05-06

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes.

Evidence of accelerated evolution and ectodermal-specific expression of presumptive BDS toxin cDNAs from Anemonia viridis.

PubMed

Nicosia, Aldo; Maggio, Teresa; Mazzola, Salvatore; Cuttitta, Angela

2013-10-30

Anemonia viridis is a widespread and extensively studied Mediterranean species of sea anemone from which a large number of polypeptide toxins, such as blood depressing substances (BDS) peptides, have been isolated. The first members of this class, BDS-1 and BDS-2, are polypeptides belonging to the β-defensin fold family and were initially described for their antihypertensive and antiviral activities. BDS-1 and BDS-2 are 43 amino acid peptides characterised by three disulfide bonds that act as neurotoxins affecting Kv3.1, Kv3.2 and Kv3.4 channel gating kinetics. In addition, BDS-1 inactivates the Nav1.7 and Nav1.3 channels. The development of a large dataset of A. viridis expressed sequence tags (ESTs) and the identification of 13 putative BDS-like cDNA sequences has attracted interest, especially as scientific and diagnostic tools. A comparison of BDS cDNA sequences showed that the untranslated regions are more conserved than the protein-coding regions. Moreover, the KA/KS ratios calculated for all pairwise comparisons showed values greater than 1, suggesting mechanisms of accelerated evolution. The structures of the BDS homologs were predicted by molecular modelling. All toxins possess similar 3D structures that consist of a triple-stranded antiparallel β-sheet and an additional small antiparallel β-sheet located downstream of the cleavage/maturation site; however, the orientation of the triple-stranded β-sheet appears to differ among the toxins. To characterise the spatial expression profile of the putative BDS cDNA sequences, tissue-specific cDNA libraries, enriched for BDS transcripts, were constructed. In addition, the proper amplification of ectodermal or endodermal markers ensured the tissue specificity of each library. Sequencing randomly selected clones from each library revealed ectodermal-specific expression of ten BDS transcripts, while transcripts of BDS-8, BDS-13, BDS-14 and BDS-15 failed to be retrieved, likely due to under-representation in our cDNA libraries. The calculation of the relative abundance of BDS transcripts in the cDNA libraries revealed that BDS-1, BDS-3, BDS-4, BDS-5 and BDS-6 are the most represented transcripts.

A computational docking study on the pH dependence of peptide binding to HLA-B27 sub-types differentially associated with ankylosing spondylitis

NASA Astrophysics Data System (ADS)

Serçinoğlu, Onur; Özcan, Gülin; Kabaş, Zeynep Kutlu; Ozbek, Pemra

2016-07-01

A single amino acid difference (Asp116His), having a key role in a pathogenesis pathway, distinguishes HLA-B*27:05 and HLA-B*27:09 sub-types as associated and non-associated with ankylosing spondylitis, respectively. In this study, molecular docking simulations were carried out with the aim of comprehending the differences in the binding behavior of both alleles at varying pH conditions. A library of modeled peptides was formed upon single point mutations aiming to address the effect of 20 naturally occurring amino acids at the binding core peptide positions. For both alleles, computational docking was applied using Autodock 4.2. Obtained free energies of binding (FEB) were compared within the peptide library and between the alleles at varying pH conditions. The amino acid preferences of each position were studied enlightening the role of each on binding. The preferred amino acids for each position of pVIPR were found to be harmonious with experimental studies. Our results indicate that, as the pH is lowered, the capacity of HLA-B*27:05 to bind peptides in the library is largely lost. Hydrogen bonding analysis suggests that the interaction between the main anchor positions of pVIPR and their respective binding pocket residues are affected from the pH the most, causing an overall shift in the FEB profiles.

A computational docking study on the pH dependence of peptide binding to HLA-B27 sub-types differentially associated with ankylosing spondylitis.

PubMed

Serçinoğlu, Onur; Özcan, Gülin; Kabaş, Zeynep Kutlu; Ozbek, Pemra

2016-07-01

A single amino acid difference (Asp116His), having a key role in a pathogenesis pathway, distinguishes HLA-B*27:05 and HLA-B*27:09 sub-types as associated and non-associated with ankylosing spondylitis, respectively. In this study, molecular docking simulations were carried out with the aim of comprehending the differences in the binding behavior of both alleles at varying pH conditions. A library of modeled peptides was formed upon single point mutations aiming to address the effect of 20 naturally occurring amino acids at the binding core peptide positions. For both alleles, computational docking was applied using Autodock 4.2. Obtained free energies of binding (FEB) were compared within the peptide library and between the alleles at varying pH conditions. The amino acid preferences of each position were studied enlightening the role of each on binding. The preferred amino acids for each position of pVIPR were found to be harmonious with experimental studies. Our results indicate that, as the pH is lowered, the capacity of HLA-B*27:05 to bind peptides in the library is largely lost. Hydrogen bonding analysis suggests that the interaction between the main anchor positions of pVIPR and their respective binding pocket residues are affected from the pH the most, causing an overall shift in the FEB profiles.

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. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Scaffold optimization in discontinuous epitope containing protein mimics of gp120 using smart libraries.

PubMed

Mulder, Gwenn E; Quarles van Ufford, H Linda C; van Ameijde, Jeroen; Brouwer, Arwin J; Kruijtzer, John A W; Liskamp, Rob M J

2013-04-28

A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC50 value of 1.7 μM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.

Interaction Analysis through Proteomic Phage Display

PubMed Central

2014-01-01

Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249

Phage display peptide libraries in molecular allergology: from epitope mapping to mimotope-based immunotherapy.

PubMed

Luzar, J; Štrukelj, B; Lunder, M

2016-11-01

Identification of allergen epitopes is a key component in proper understanding of the pathogenesis of type I allergies, for understanding cross-reactivity and for the development of mimotope immunotherapeutics. Phage particles have garnered recognition in the field of molecular allergology due to their value not only in competitive immunoscreening of peptide libraries but also as immunogenic carriers of allergen mimotopes. They integrate epitope discovery technology and immunization functions into a single platform. This article provides an overview of allergen mimotopes identified through the phage display technique. We discuss the contribution of phage display peptide libraries in determining dominant B-cell epitopes of allergens, in developing mimotope immunotherapy, in understanding cross-reactivity, and in determining IgE epitope profiles of individual patients to improve diagnostics and individualize immunotherapy. We also discuss the advantages and pitfalls of the methodology used to identify and validate the mimotopes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

PubMed

Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

2015-03-23

Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

Single-round selection yields a unique retroviral envelope utilizing GPR172A as its host receptor.

PubMed

Mazari, Peter M; Linder-Basso, Daniela; Sarangi, Anindita; Chang, Yehchung; Roth, Monica J

2009-04-07

The recognition by a viral envelope of its cognate host-cell receptor is the initial critical step in defining the viral host-range and tissue specificity. This study combines a single-round of selection of a random envelope library with a parallel cDNA screen for receptor function to identify a distinct retroviral envelope/receptor pair. The 11-aa targeting domain of the modified feline leukemia virus envelope consists of a constrained peptide. Critical to the binding of the constrained peptide envelope to its cellular receptor are a pair of internal cysteines and an essential Trp required for maintenance of titers >10(5) lacZ staining units per milliliter. The receptor used for viral entry is the human GPR172A protein, a G-protein-coupled receptor isolated from osteosarcoma cells. The ability to generate unique envelopes capable of using tissue- or disease-specific receptors marks an advance in the development of efficient gene-therapy vectors.

Phage display for the discovery of hydroxyapatite-associated peptides.

PubMed

Jin, Hyo-Eon; Chung, Woo-Jae; Lee, Seung-Wuk

2013-01-01

In nature, proteins play a critical role in the biomineralization process. Understanding how different peptide or protein sequences selectively interact with the target crystal is of great importance. Identifying such protein structures is one of the critical steps in verifying the molecular mechanisms of biomineralization. One of the promising ways to obtain such information for a particular crystal surface is to screen combinatorial peptide libraries in a high-throughput manner. Among the many combinatorial library screening procedures, phage display is a powerful method to isolate such proteins and peptides. In this chapter, we will describe our established methods to perform phage display with inorganic crystal surfaces. Specifically, we will use hydroxyapatite as a model system for discovery of apatite-associated proteins in bone or tooth biomineralization studies. This model approach can be generalized to other desired crystal surfaces using the same experimental design principles with a little modification of the procedures. © 2013 Elsevier Inc. All rights reserved.

Combination of Multiple Spectral Libraries Improves the Current Search Methods Used to Identify Missing Proteins in the Chromosome-Centric Human Proteome Project.

PubMed

Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Kim, Kwang-Youl; Kwon, Kyung-Hoon; Yoo, Jong Shin; Omenn, Gilbert S; Baker, Mark S; Hancock, William S; Paik, Young-Ki

2015-12-04

Approximately 2.9 billion long base-pair human reference genome sequences are known to encode some 20 000 representative proteins. However, 3000 proteins, that is, ~15% of all proteins, have no or very weak proteomic evidence and are still missing. Missing proteins may be present in rare samples in very low abundance or be only temporarily expressed, causing problems in their detection and protein profiling. In particular, some technical limitations cause missing proteins to remain unassigned. For example, current mass spectrometry techniques have high limits and error rates for the detection of complex biological samples. An insufficient proteome coverage in a reference sequence database and spectral library also raises major issues. Thus, the development of a better strategy that results in greater sensitivity and accuracy in the search for missing proteins is necessary. To this end, we used a new strategy, which combines a reference spectral library search and a simulated spectral library search, to identify missing proteins. We built the human iRefSPL, which contains the original human reference spectral library and additional peptide sequence-spectrum match entries from other species. We also constructed the human simSPL, which contains the simulated spectra of 173 907 human tryptic peptides determined by MassAnalyzer (version 2.3.1). To prove the enhanced analytical performance of the combination of the human iRefSPL and simSPL methods for the identification of missing proteins, we attempted to reanalyze the placental tissue data set (PXD000754). The data from each experiment were analyzed using PeptideProphet, and the results were combined using iProphet. For the quality control, we applied the class-specific false-discovery rate filtering method. All of the results were filtered at a false-discovery rate of <1% at the peptide and protein levels. The quality-controlled results were then cross-checked with the neXtProt DB (2014-09-19 release). The two spectral libraries, iRefSPL and simSPL, were designed to ensure no overlap of the proteome coverage. They were shown to be complementary to spectral library searching and significantly increased the number of matches. From this trial, 12 new missing proteins were identified that passed the following criterion: at least 2 peptides of 7 or more amino acids in length or one of 9 or more amino acids in length with one or more unique sequences. Thus, the iRefSPL and simSPL combination can be used to help identify peptides that have not been detected by conventional sequence database searches with improved sensitivity and a low error rate.

Efficacy of positive airway pressure on brain natriuretic peptide in patients with heart failure and sleep-disorder breathing: a meta-analysis of randomized controlled trials.

PubMed

Zhang, Xiao-Bin; Yuan, Ya-Ting; Du, Yan-Ping; Jiang, Xing-Tang; Zeng, Hui-Qing

2015-04-01

Positive airway pressure (PAP) has been recognized as an effective therapeutic option for sleep-disordered breathing (SDB) in patients with heart failure (HF), and it can improve left ventricular function. Whether PAP can ameliorate serum brain natriuretic peptide (BNP) levels, a biomarker of HF, is controversial. The purpose of the present study was to quantitatively assess the efficacy of PAP on BNP in patients with HF and SDB. A systematic search of PubMed, Embase, Web of Science and Cochrane library identified six randomized controlled trials (RCTs), in which PAP was compared with medical therapy, subtherapeutic PAP or different types of PAP. The data of BNP were extracted and pooled into meta-analysis using STATA 12.0. Totally 6 RCT studies (7 cohorts) with 222 patients were enrolled into analysis. The quality of each study was high and the heterogeneity (I(2) = 58.1%) was noted between studies. A significant reduction of BNP was observed after PAP treatment in patients with HF and SDB (SMD -0.517, 95% CI -0.764 to -0.270, z = 4.11, p = 0.000). Our meta-analysis of RCTs demonstrated that PAP elicits significant reduction of BNP in patients with HF and SDB.

Enhanced Sensitive Immunoassay: Noncompetitive Phage Anti-Immune Complex Assay for the Determination of Malachite Green and Leucomalachite Green

PubMed Central

2015-01-01

To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG–mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R2LMG = 0.9841; R2MG = 0.993; R2Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety. PMID:25077381

Biased selection of propagation-related TUPs from phage display peptide libraries.

PubMed

Zade, Hesam Motaleb; Keshavarz, Reihaneh; Shekarabi, Hosna Sadat Zahed; Bakhshinejad, Babak

2017-08-01

Phage display is rapidly advancing as a screening strategy in drug discovery and drug delivery. Phage-encoded combinatorial peptide libraries can be screened through the affinity selection procedure of biopanning to find pharmaceutically relevant cell-specific ligands. However, the unwanted enrichment of target-unrelated peptides (TUPs) with no true affinity for the target presents an important barrier to the successful screening of phage display libraries. Propagation-related TUPs (Pr-TUPs) are an emerging but less-studied category of phage display-derived false-positive hits that are displayed on the surface of clones with faster propagation rates. Despite long regarded as an unbiased selection system, accumulating evidence suggests that biopanning may create biological bias toward selection of phage clones with certain displayed peptides. This bias can be dependent on or independent of the displayed sequence and may act as a major driving force for the isolation of fast-growing clones. Sequence-dependent bias is reflected by censorship or over-representation of some amino acids in the displayed peptide and sequence-independent bias is derived from either point mutations or rare recombination events occurring in the phage genome. It is of utmost interest to clean biopanning data by identifying and removing Pr-TUPs. Experimental and bioinformatic approaches can be exploited for Pr-TUP discovery. With no doubt, obtaining deeper insight into how Pr-TUPs emerge during biopanning and how they could be detected provides a basis for using cell-targeting peptides isolated from phage display screening in the development of disease-specific diagnostic and therapeutic platforms.

Mimtags: the use of phage display technology to produce novel protein-specific probes.

PubMed

Ahmed, Nayyar; Dhanapala, Pathum; Sadli, Nadia; Barrow, Colin J; Suphioglu, Cenk

2014-03-01

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

Peptidomics combined with cDNA library unravel the diversity of centipede venom.

PubMed

Rong, Mingqiang; Yang, Shilong; Wen, Bo; Mo, Guoxiang; Kang, Di; Liu, Jie; Lin, Zhilong; Jiang, Wenbin; Li, Bowen; Du, Chaoqin; Yang, Shuanjuan; Jiang, Hui; Feng, Qiang; Xu, Xun; Wang, Jun; Lai, Ren

2015-01-30

Centipedes are one of the oldest venomous arthropods using toxin as their weapon to capture prey. But little attention was focused on them and only few centipede toxins were demonstrated with activity on ion channels. Therefore, more deep works are needed to understand the diversity of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins were more complicated than any other animal toxins and even exhibited large differences in homologues. Meanwhile, a large number of variants generated by alternative cleavage sites were detected by mass spectra. Odd number of cystein (3, 5, 7) found in the mature peptides were seldom seen in peptide toxins. In additional, two novel cysteine frameworks (C-C-C-CCC, C-C-C-C-CC-CC) were identified from 16 different cysteine frameworks from centipede peptides. Only 29 precursors have clear targets, while others may provide a potential diversity function for centipede. These findings highlight the extensive diversity of centipede toxins and provide powerful tools to understand the capture and defense weapon of centipede. Peptide toxins from venomous animal have attracted increasing attentions due to their extraordinary chemical and pharmacological diversity. Centipedes are one of the most used Chinese traditional medicines, but little was known about the active components. The venom of Scolopendra subspinipes mutilans L. Koch is first deeply analyzed in this work and most of peptides were never discovered before. Interestingly, the number and arrangement of cysteine showed a larger different to known peptide toxins such spider or scorpion toxins. Moreover, only 29 peptides from this centipede venom were identified with known function. It suggested that our work not only important to understand the composition of centipede venom, but also provide many valuable peptides for potential biological functions. Copyright © 2014 Elsevier B.V. All rights reserved.

Using the ribosome to synthesize peptidomimetics

PubMed Central

2009-01-01

Peptidomimetic research is an approach to identify peptide-based drugs designed to mimic structural, conformational, and biological properties of peptides while overcoming their limitations, such as protease instability and poor cell penetration. With recent advances in ribosomal synthesis of peptides containing unnatural amino acids, this technology appears suitable for preparing large structurally diverse libraries of peptidomimetics for drug discovery screening. PMID:20948631

Advances in Synthetic Peptides Reagent Discovery

DTIC Science & Technology

2013-07-01

to promote specific and high affinity binding. Longer incubations may result in nonspecific attachment, such as early biofilm formation. Because...peptide libraries yields ligand arrays that classify breast tumor subtypes,” Molecular Cancer Therapeutics, 8(5), 1312-1318 (2009). [26] J. M. Kogot

De Novo Design of Skin-Penetrating Peptides for Enhanced Transdermal Delivery of Peptide Drugs.

PubMed

Menegatti, Stefano; Zakrewsky, Michael; Kumar, Sunny; De Oliveira, Joshua Sanchez; Muraski, John A; Mitragotri, Samir

2016-03-09

Skin-penetrating peptides (SPPs) are attracting increasing attention as a non-invasive strategy for transdermal delivery of therapeutics. The identification of SPP sequences, however, currently performed by experimental screening of peptide libraries, is very laborious. Recent studies have shown that, to be effective enhancers, SPPs must possess affinity for both skin keratin and the drug of interest. We therefore developed a computational process for generating and screening virtual libraries of disulfide-cyclic peptides against keratin and cyclosporine A (CsA) to identify SPPs capable of enhancing transdermal CsA delivery. The selected sequences were experimentally tested and found to bind both CsA and keratin, as determined by mass spectrometry and affinity chromatography, and enhance transdermal permeation of CsA. Four heptameric sequences that emerged as leading candidates (ACSATLQHSCG, ACSLTVNWNCG, ACTSTGRNACG, and ACSASTNHNCG) were tested and yielded CsA permeation on par with previously identified SPP SPACE (TM) . An octameric peptide (ACNAHQARSTCG) yielded significantly higher delivery of CsA compared to heptameric SPPs. The safety profile of the selected sequences was also validated by incubation with skin keratinocytes. This method thus represents an effective procedure for the de novo design of skin-penetrating peptides for the delivery of desired therapeutic or cosmetic agents. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tailoring gas sensor arrays via the design of short peptides sequences as binding elements.

PubMed

Mascini, Marcello; Pizzoni, Daniel; Perez, German; Chiarappa, Emilio; Di Natale, Corrado; Pittia, Paola; Compagnone, Dario

2017-07-15

A semi-combinatorial virtual approach was used to prepare peptide-based gas sensors with binding properties towards five different chemical classes (alcohols, aldehydes, esters, hydrocarbons and ketones). Molecular docking simulations were conducted for a complete tripeptide library (8000 elements) versus 58 volatile compounds belonging to those five chemical classes. By maximizing the differences between chemical classes, a subset of 120 tripeptides was extracted and used as scaffolds for generating a combinatorial library of 7912 tetrapeptides. This library was processed in an analogous way to the former. Five tetrapeptides (IHRI, KSDS, LGFD, TGKF and WHVS) were chosen depending on their virtual affinity and cross-reactivity for the experimental step. The five peptides were covalently bound to gold nanoparticles by adding a terminal cysteine to each tetrapeptide and deposited onto 20MHz quartz crystal microbalances to construct the gas sensors. The behavior of peptides after this chemical modification was simulated at the pH range used in the immobilization step. ΔF signals analyzed by principal component analysis matched the virtually screened data. The array was able to clearly discriminate the 13 volatile compounds tested based on their hydrophobicity and hydrophilicity molecules as well as the molecular weight. Copyright © 2016 Elsevier B.V. All rights reserved.

Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

PubMed

Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

2014-08-01

Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

Extensive reprogramming of the genetic code for genetically encoded synthesis of highly N-alkylated polycyclic peptidomimetics.

PubMed

Kawakami, Takashi; Ishizawa, Takahiro; Murakami, Hiroshi

2013-08-21

Cyclic structures can increase the proteolytic stability and conformational rigidity of peptides, and N-alkylation of the peptide backbone can make peptides more cell-permeable and resistant to proteolysis. Therefore, cyclic N-alkyl amino acids are expected to be useful building blocks to increase simultaneously these pharmacological properties of peptides. In this study, we screened various cyclic N-alkyl amino acids for their ribosomal incorporation into peptides and identified cyclic N-alkyl amino acids that can be efficiently and successively incorporated. We also demonstrated genetic code reprogramming for reassigning 16 NNU codons to 16 different cyclic N-alkyl amino acids with high fidelity to synthesize highly N-alkylated polycyclic peptidomimetics and an mRNA-displayed library of completely N-alkylated polycyclic peptidomimetics by using our recently developed TRAP (transcription/translation coupled with association of puromycin linker) display. In vitro selection from a highly diverse library of such completely N-alkylated polycyclic peptidomimetics could become a powerful means to discover small-molecule ligands such as drug candidates that can be targeted to biomolecules inside living cells.

Identification and characterization of a peptide affinity reagent for detection of noroviruses in clinical samples.

PubMed

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

2013-06-01

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.

A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity.

PubMed

Lindberg, Hanna; Härd, Torleif; Löfblom, John; Ståhl, Stefan

2015-09-01

The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide. © 2015 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-Non-Commercial-NoDerivs Licence, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Sialome of a Generalist Lepidopteran Herbivore: Identification of Transcripts and Proteins from Helicoverpa armigera Labial Salivary Glands

PubMed Central

Celorio-Mancera, Maria de la Paz; Courtiade, Juliette; Muck, Alexander; Heckel, David G.; Musser, Richard O.; Vogel, Heiko

2011-01-01

Although the importance of insect saliva in insect-host plant interactions has been acknowledged, there is very limited information on the nature and complexity of the salivary proteome in lepidopteran herbivores. We inspected the labial salivary transcriptome and proteome of Helicoverpa armigera, an important polyphagous pest species. To identify the majority of the salivary proteins we have randomly sequenced 19,389 expressed sequence tags (ESTs) from a normalized cDNA library of salivary glands. In parallel, a non-cytosolic enriched protein fraction was obtained from labial salivary glands and subjected to two-dimensional gel electrophoresis (2-DE) and de novo peptide sequencing. This procedure allowed comparison of peptides and EST sequences and enabled us to identify 65 protein spots from the secreted labial saliva 2DE proteome. The mass spectrometry analysis revealed ecdysone, glucose oxidase, fructosidase, carboxyl/cholinesterase and an uncharacterized protein previously detected in H. armigera midgut proteome. Consistently, their corresponding transcripts are among the most abundant in our cDNA library. We did find redundancy of sequence identification of saliva-secreted proteins suggesting multiple isoforms. As expected, we found several enzymes responsible for digestion and plant offense. In addition, we identified non-digestive proteins such as an arginine kinase and abundant proteins of unknown function. This identification of secreted salivary gland proteins allows a more comprehensive understanding of insect feeding and poses new challenges for the elucidation of protein function. PMID:22046331

Complementary DNA libraries: an overview.

PubMed

Ying, Shao-Yao

2004-07-01

The generation of complete and full-length cDNA libraries for potential functional assays of specific gene sequences is essential for most molecules in biotechnology and biomedical research. The field of cDNA library generation has changed rapidly in the past 10 yr. This review presents an overview of the method available for the basic information of generating cDNA libraries, including the definition of the cDNA library, different kinds of cDNA libraries, difference between methods for cDNA library generation using conventional approaches and a novel strategy, and the quality of cDNA libraries. It is anticipated that the high-quality cDNA libraries so generated would facilitate studies involving genechips and the microarray, differential display, subtractive hybridization, gene cloning, and peptide library generation.

SAR by Oxime-Containing Peptide Libraries: Application to Tsg101 Ligand Optimization

PubMed Central

Liu, Fa; Stephen, Andrew G.; Waheed, Abdul A.; Aman, M. Javad; Freed, Eric O.; Fisher, Robert J.; Burke, Terrence R.

2008-01-01

HIV-1 viral assembly requires a direct interaction between a Pro-Thr-Ala-Pro (“PTAP”) motif in the viral protein Gag-p6 and the cellular endosomal sorting factor Tsg101. In an effort to develop competitive inhibitors of this interaction, an SAR study was conducted based on the application of post solid-phase oxime formation involving the sequential insertion of aminooxy-containing residues within a nonamer parent peptide followed by reaction with libraries of aldehydes. Approximately 15–20-fold enhancement in binding affinity was achieved by this approach. PMID:18655064

MRM screening/biomarker discovery with linear ion trap MS: a library of human cancer-specific peptides.

PubMed

Yang, Xu; Lazar, Iulia M

2009-03-27

The discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS)-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM), have been implemented. MCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX)/reversed phase liquid chromatography (RPLC) separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron). In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide. In this work, we report on the generation of a library of 9,677 peptides (p < 0.001), representing approximately 1,572 proteins from human breast cancer cells, that can be used for MRM/MS-based biomarker screening studies. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum (p-value, DeltaM, Xcorr, DeltaCn, Sp, no. of matching a, b, y ions in the spectrum), the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW)<50,000. Preliminary experiments have demonstrated that putative biomarkers, that are not detectable by conventional data dependent MS acquisition methods in complex un-fractionated samples, can be reliable identified with the information provided in this library. Based on the spectral count, the quality of a tandem mass spectrum and the m/z values for a parent peptide and its most abundant daughter ions, MRM conditions can be selected to enable the detection of target peptides and proteins.

MRM screening/biomarker discovery with linear ion trap MS: a library of human cancer-specific peptides

PubMed Central

2009-01-01

Background The discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS)-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM), have been implemented. Methods MCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX)/reversed phase liquid chromatography (RPLC) separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron). In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide. Results In this work, we report on the generation of a library of 9,677 peptides (p < 0.001), representing ~1,572 proteins from human breast cancer cells, that can be used for MRM/MS-based biomarker screening studies. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum (p-value, DeltaM, Xcorr, DeltaCn, Sp, no. of matching a, b, y ions in the spectrum), the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW)<50,000. Conclusion Preliminary experiments have demonstrated that putative biomarkers, that are not detectable by conventional data dependent MS acquisition methods in complex un-fractionated samples, can be reliable identified with the information provided in this library. Based on the spectral count, the quality of a tandem mass spectrum and the m/z values for a parent peptide and its most abundant daughter ions, MRM conditions can be selected to enable the detection of target peptides and proteins. PMID:19327145

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

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

Bioassembled layered silicate-metal nanoparticle hybrids.

PubMed

Drummy, Lawrence F; Jones, Sharon E; Pandey, Ras B; Farmer, B L; Vaia, Richard A; Naik, Rajesh R

2010-05-01

Here we report on the bioenabled assembly of layered nanohybrids using peptides identified with regard to their affinity to the nanoparticle surface. A dodecamer peptide termed M1, determined from a phage peptide display library, was found to bind to the surface of a layered aluminosilicate (montmorillonite, MMT). Fusion of a metal binding domain to the M1 peptide or the M1 peptide by itself was able to direct the growth of metal nanoparticles, such as gold and cobalt-platinum, respectively, on the MMT. This method of producing hybrid nanoclay materials will have utility in catalytic, optical, biomedical, and composite materials applications.

Preparation of human Melanocortin-4 receptor agonist libraries: linear peptides X-Y-DPhe7-Arg8-Trp(or 2-Nal)9-Z-NH2.

PubMed

Cheung, Adrian Wai-Hing; Qi, Lida; Gore, Vijay; Chu, Xin-Jie; Bartkovitz, David; Kurylko, Grazyna; Swistok, Joseph; Danho, Waleed; Chen, Li; Yagaloff, Keith

2005-12-15

Two libraries of hMC4R agonists, X-Y-DPhe(7)-Arg(8)-2-Nal(9)-Z-NH(2) and X-Y-DPhe(7)-Arg(8)-Trp(9)-Z-NH(2), totaling 185 peptides were prepared using Irori radiofrequency tagging technology and Argonaut Quest 210 Synthesizer, where X stands for N-caps, Y for His(6) surrogates and Z for Gly(10) surrogates. As a result of this study, His-modified pentapeptides with Trp were found to be more hMC4R potent than the corresponding 2-Nal analogs, novel N-caps and Gly surrogates were identified and 19 new peptides which are potent hMC4R agonists (EC(50) 1-15nM) and selective against hMC1R were discovered.

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.

A minimal peptide scaffold for beta-turn display: optimizing a strand position in disulfide-cyclized beta-hairpins.

PubMed

Cochran, A G; Tong, R T; Starovasnik, M A; Park, E J; McDowell, R S; Theaker, J E; Skelton, N J

2001-01-31

Phage display of peptide libraries has become a powerful tool for the evolution of novel ligands that bind virtually any protein target. However, the rules governing conformational preferences in natural peptides are poorly understood, and consequently, structure-activity relationships in these molecules can be difficult to define. In an effort to simplify this process, we have investigated the structural stability of 10-residue, disulfide-constrained beta-hairpins and assessed their suitability as scaffolds for beta-turn display. Using disulfide formation as a probe, relative free energies of folding were measured for 19 peptides that differ at a one strand position. A tryptophan substitution promotes folding to a remarkable degree. NMR analysis confirms that the measured energies correlate well with the degree of beta-hairpin structure in the disulfide-cyclized peptides. Reexamination of a subset of the strand substitutions in peptides with different turn sequences reveals linear free energy relationships, indicating that turns and strand-strand interactions make independent, additive contributions to hairpin stability. Significantly, the tryptophan strand substitution is highly stabilizing with all turns tested, and peptides that display model turns or the less stable C'-C' ' turn of CD4 on this tryptophan "stem" are highly structured beta-hairpins in water. Thus, we have developed a small, structured beta-turn scaffold, containing only natural L-amino acids, that may be used to display peptide libraries of limited conformational diversity on phage.

Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2

PubMed Central

Keresztessy, Zsolt; Csősz, Éva; Hársfalvi, Jolán; Csomós, Krisztián; Gray, Joe; Lightowlers, Robert N.; Lakey, Jeremy H.; Balajthy, Zoltán; Fésüs, László

2006-01-01

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q6, Q8, and Q22 are modified by TG2. Kinetic parameters of SnQ1 transamidation (KMapp = 250 μM, kcat = 18.3 sec−1, and kcat/KMapp = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research. PMID:17075129

Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2.

PubMed

Keresztessy, Zsolt; Csosz, Eva; Hársfalvi, Jolán; Csomós, Krisztián; Gray, Joe; Lightowlers, Robert N; Lakey, Jeremy H; Balajthy, Zoltán; Fésüs, László

2006-11-01

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q(6), Q(8), and Q(22) are modified by TG2. Kinetic parameters of SnQ1 transamidation (K(M)(app) = 250 microM, k(cat) = 18.3 sec(-1), and k(cat)/K(M)(app) = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.

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.

Limnonectins: a new class of antimicrobial peptides from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis).

PubMed

Wu, Youjia; Wang, Lei; Zhou, Mei; Ma, Chengbang; Chen, Xiaole; Bai, Bing; Chen, Tianbao; Shaw, Chris

2011-06-01

Amphibian skin secretions are rich sources of biologically-active peptides with antimicrobial peptides predominating in many species. Several studies involving molecular cloning of biosynthetic precursor-encoding cDNAs from skin or skin secretions have revealed that these exhibit highly-conserved domain architectures with an unusually high degree of conserved nucleotide and resultant amino acid sequences within the signal peptides. This high degree of nucleotide sequence conservation has permitted the design of primers complementary to such sites facilitating "shotgun" cloning of skin or skin secretion-derived cDNA libraries from hitherto unstudied species. Here we have used such an approach using a skin secretion-derived cDNA library from an unstudied species of Chinese frog - the Fujian large-headed frog, Limnonectes fujianensis - and have discovered two 16-mer peptides of novel primary structures, named limnonectin-1Fa (SFPFFPPGICKRLKRC) and limnonectin-1Fb (SFHVFPPWMCKSLKKC), that represent the prototypes of a new class of amphibian skin antimicrobial peptide. Unusually these limnonectins display activity only against a Gram-negative bacterium (MICs of 35 and 70 μM) and are devoid of haemolytic activity at concentrations up to 160 μM. Thus the "shotgun" cloning approach described can exploit the unusually high degree of nucleotide conservation in signal peptide-encoding domains of amphibian defensive skin secretion peptide precursor-encoding cDNAs to rapidly expedite the discovery of novel and functional defensive peptides in a manner that circumvents specimen sacrifice without compromising robustness of data. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

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 to mimic the known endogenous ligands. These efforts led to the discovery of high-affinity α-helix mimetics (K(i) = 0.7 μM) against HIV-1 gp41 as well as high-affinity and selective β-turn mimetics (K(i) = 80 nM) against the κ-opioid receptor. The results suggest that the use of such comprehensive libraries of peptide secondary structure mimetics, built around effective molecular scaffolds, constitutes a powerful method of interrogating PPIs. These structures provide small molecule modulators of PPI networks for therapeutic target validation, lead compound discovery, and the identification of modulators of biological processes for further study.

Evaluation of proteases and protease inhibitors in Heterodera glycines cysts obtained from laboratory and field populations

USDA-ARS?s Scientific Manuscript database

Proteases and proteases inhibitors were evaluated in a number of preparations of Heterodera glycines cysts obtained from glasshouse cultures (GH) and field (LR) populations. Using a FRET-peptide library comprising 512 peptide substrate pools that detect 4 endoprotease types (aspartic, cysteine, meta...

Biomining of MoS2 with Peptide-based Smart Biomaterials.

PubMed

Cetinel, Sibel; Shen, Wei-Zheng; Aminpour, Maral; Bhomkar, Prasanna; Wang, Feng; Borujeny, Elham Rafie; Sharma, Kumakshi; Nayebi, Niloofar; Montemagno, Carlo

2018-02-20

Biomining of valuable metals using a target specific approach promises increased purification yields and decreased cost. Target specificity can be implemented with proteins/peptides, the biological molecules, responsible from various structural and functional pathways in living organisms by virtue of their specific recognition abilities towards both organic and inorganic materials. Phage display libraries are used to identify peptide biomolecules capable of specifically recognizing and binding organic/inorganic materials of interest with high affinities. Using combinatorial approaches, these molecular recognition elements can be converted into smart hybrid biomaterials and harnessed for biotechnological applications. Herein, we used a commercially available phage-display library to identify peptides with specific binding affinity to molybdenite (MoS 2 ) and used them to decorate magnetic NPs. These peptide-coupled NPs could capture MoS 2 under a variety of environmental conditions. The same batch of NPs could be re-used multiple times to harvest MoS 2 , clearly suggesting that this hybrid material was robust and recyclable. The advantages of this smart hybrid biomaterial with respect to its MoS 2 -binding specificity, robust performance under environmentally challenging conditions and its recyclability suggests its potential application in harvesting MoS 2 from tailing ponds and downstream mining processes.

Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of factor VIII.

PubMed

Kelley, Brian D; Tannatt, Molly; Magnusson, Robert; Hagelberg, Sigrid; Booth, James

2004-08-05

An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of approximately 1 microM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3-4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials.

In Vivo 18 FDG/18 Choline Mediated Cerenkov Radiation Energy Transfer (CRET) Multiplexed Optical Imaging for Human Prostate Carcinoma Detection and Staging

DTIC Science & Technology

2016-10-01

human prostate cancer xenografts. We have selected peptides from bacteriophage display libraries that target TF and ErbB2/ErbB3. The peptides have been...facilitate biomarker-specific diagnosis. The specific aims of the proposal are to: 1) select peptides that target the ErbB2/3 heterodimer using novel...parallel in vitro/in vivo phage display techniques; 2) generate NIR-QDs decorated with TF- and ErbB2/3-avid peptides for in vivo molecular

Microbeads display of proteins using emulsion PCR and cell-free protein synthesis.

PubMed

Gan, Rui; Yamanaka, Yumiko; Kojima, Takaaki; Nakano, Hideo

2008-01-01

We developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell-free protein synthesis in emulsion. A PCR mixture containing streptavidin-coated microbeads was compartmentalized by water-in-oil (w/o) emulsion with estimated 0.5 template molecules per droplet. The template molecules were amplified and immobilized on beads via bead-linked reverse primers and biotinylated forward primers. After amplification, the templates were sequentially labeled with streptavidin and biotinylated anti-glutathione S-transferase (GST) antibody. The pool of beads was then subjected to cell-free protein synthesis compartmentalized in another w/o emulsion, in which templates were coupled to their coding proteins. We mixed two types of DNA templates of Histidine6 tag (His6)-fused and FLAG tag-fused GST in a ratio of 1:1,000 (His6: FLAG) for use as a model DNA library. After incubation with fluorescein isothiocyanate (FITC)-labeled anti-His6 (C-term) antibody, the beads with the His6 gene were enriched 917-fold in a single-round screening by using flow cytometry. A library with a theoretical diversity of 10(6) was constructed by randomizing the middle four residues of the His6 tag. After a two-round screening, the randomized sequences were substantially converged to peptide-encoding sequences recognized by the anti-His6 antibody.

Designing of interferon-gamma inducing MHC class-II binders

PubMed Central

2013-01-01

Background The generation of interferon-gamma (IFN-γ) by MHC class II activated CD4+ T helper cells play a substantial contribution in the control of infections such as caused by Mycobacterium tuberculosis. In the past, numerous methods have been developed for predicting MHC class II binders that can activate T-helper cells. Best of author’s knowledge, no method has been developed so far that can predict the type of cytokine will be secreted by these MHC Class II binders or T-helper epitopes. In this study, an attempt has been made to predict the IFN-γ inducing peptides. The main dataset used in this study contains 3705 IFN-γ inducing and 6728 non-IFN-γ inducing MHC class II binders. Another dataset called IFNgOnly contains 4483 IFN-γ inducing epitopes and 2160 epitopes that induce other cytokine except IFN-γ. In addition we have alternate dataset that contains IFN-γ inducing and equal number of random peptides. Results It was observed that the peptide length, positional conservation of residues and amino acid composition affects IFN-γ inducing capabilities of these peptides. We identified the motifs in IFN-γ inducing binders/peptides using MERCI software. Our analysis indicates that IFN-γ inducing and non-inducing peptides can be discriminated using above features. We developed models for predicting IFN-γ inducing peptides using various approaches like machine learning technique, motifs-based search, and hybrid approach. Our best model based on the hybrid approach achieved maximum prediction accuracy of 82.10% with MCC of 0.62 on main dataset. We also developed hybrid model on IFNgOnly dataset and achieved maximum accuracy of 81.39% with 0.57 MCC. Conclusion Based on this study, we have developed a webserver for predicting i) IFN-γ inducing peptides, ii) virtual screening of peptide libraries and iii) identification of IFN-γ inducing regions in antigen (http://crdd.osdd.net/raghava/ifnepitope/). Reviewers This article was reviewed by Prof Kurt Blaser, Prof Laurence Eisenlohr and Dr Manabu Sugai. PMID:24304645

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.

Thermo-msf-parser: an open source Java library to parse and visualize Thermo Proteome Discoverer msf files.

PubMed

Colaert, Niklaas; Barsnes, Harald; Vaudel, Marc; Helsens, Kenny; Timmerman, Evy; Sickmann, Albert; Gevaert, Kris; Martens, Lennart

2011-08-05

The Thermo Proteome Discoverer program integrates both peptide identification and quantification into a single workflow for peptide-centric proteomics. Furthermore, its close integration with Thermo mass spectrometers has made it increasingly popular in the field. Here, we present a Java library to parse the msf files that constitute the output of Proteome Discoverer. The parser is also implemented as a graphical user interface allowing convenient access to the information found in the msf files, and in Rover, a program to analyze and validate quantitative proteomics information. All code, binaries, and documentation is freely available at http://thermo-msf-parser.googlecode.com.

Combinatorially Screened Peptide as Targeted Covalent Binder: Alteration of Bait-Conjugated Peptide to Reactive Modifier.

PubMed

Uematsu, Shuta; Tabuchi, Yudai; Ito, Yuji; Taki, Masumi

2018-06-01

A peptide-type covalent binder for a target protein was obtained by combinatorial screening of fluoroprobe-conjugated peptide libraries on bacteriophage T7. The solvatochromic fluoroprobe works as a bait during the affinity selection process of phage display. To obtain the targeted covalent binder, the bait in the selected consensus peptide was altered into a reactive warhead possessing a sulfonyl fluoride. The reaction efficiency and site/position specificity of the covalent conjugation between the binder and the target protein were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and rationalized by a protein-ligand docking simulation.

Peptoids: a modular approach to drug discovery.

PubMed Central

Simon, R J; Kania, R S; Zuckermann, R N; Huebner, V D; Jewell, D A; Banville, S; Ng, S; Wang, L; Rosenberg, S; Marlowe, C K

1992-01-01

Peptoids, oligomers of N-substituted glycines, are described as a motif for the generation of chemically diverse libraries of novel molecules. Ramachandran-type plots were calculated and indicate a greater diversity of conformational states available for peptoids than for peptides. The monomers incorporate t-butyl-based side-chain and 9-fluorenylmethoxy-carbonyl alpha-amine protection. The controlled oligomerization of the peptoid monomers was performed manually and robotically with in situ activation by either benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate or bromotris(pyrrolidino)phosphonium hexaflurophosphate. Other steps were identical to peptide synthesis using alpha-(9-fluorenylmethoxycarbonyl)amino acids. A total of 15 monomers and 10 oligomers (peptoids) are described. Preliminary data are presented on the stability of a representative oligopeptoid to enzymatic hydrolysis. Peptoid versions of peptide ligands of three biological systems (bovine pancreatic alpha-amylase, hepatitis A virus 3C proteinase, and human immunodeficiency virus transactivator-responsive element RNA) were found with affinities comparable to those of the corresponding peptides. The potential use of libraries of these compounds in receptor- or enzyme-based assays is discussed. PMID:1409642

Novel Peptide Ligands of RGS4 from a Focused One-Bead, One-Compound Library

PubMed Central

Roof, Rebecca A.; Sobczyk-Kojiro, Katarzyna; Turbiak, Anjanette J.; Roman, David L.; Pogozheva, Irina D.; Blazer, Levi L.; Neubig, Richard R.; Mosberg, Henry I.

2010-01-01

Regulators of G Protein Signaling (RGS) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors. The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We previously reported the design, mechanistic evaluation and structure-activity relationships (SAR) of a disulfide-containing cyclic peptide inhibitor of RGS4, YJ34 (Ac-Val-Lys-c[Cys-Thr-Gly-Ile-Cys]-Glu-NH2, S-S) (Roof, et al. Chem Biol Drug Des 2006; 67:266-274). Using a focused one-bead, one-compound (OBOC) peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2, (Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH2, S-S with a free or acetylated N-terminus) inhibited RGS4-stimulated Gαo GTPase activity at 25–50 μM. They selectively inhibit RGS4 but not RGS7, RGS16 and RGS19. Their inhibition of RGS4 does not depend on cysteine-modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation. PMID:18637987

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes.

PubMed

Ivarsson, Ylva; Arnold, Roland; McLaughlin, Megan; Nim, Satra; Joshi, Rakesh; Ray, Debashish; Liu, Bernard; Teyra, Joan; Pawson, Tony; Moffat, Jason; Li, Shawn Shun-Cheng; Sidhu, Sachdev S; Kim, Philip M

2014-02-18

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.

High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

PubMed Central

2015-01-01

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays.

PubMed

Whiteaker, Jeffrey R; Zhao, Lei; Frisch, Christian; Ylera, Francisco; Harth, Stefan; Knappik, Achim; Paulovich, Amanda G

2014-04-04

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.

[Elaboration of Pseudo-natural Products Using Artificial In Vitro Biosynthesis Systems].

PubMed

Goto, Yuki

2018-01-01

 Peptidic natural products often consist of not only proteinogenic building blocks but also unique non-proteinogenic structures such as macrocyclic scaffolds and N-methylated backbones. Since such non-proteinogenic structures are important structural motifs that contribute to diverse bioactivity, we have proposed that peptides with non-proteinogenic structures should be attractive candidates as artificial bioactive peptides mimicking natural products, or so-called pseudo-natural products. We previously devised an engineered translation system for pseudo-natural peptides, referred to as the flexible in vitro translation (FIT) system. This system enabled "one-pot" synthesis of highly diverse pseudo-natural peptide libraries, which can be rapidly screened by mRNA display technology for the discovery of pseudo-natural peptides with diverse bioactivities.

In vitro and direct in vivo testing of mixture-based combinatorial libraries for the identification of highly active and specific opiate ligands.

PubMed

Houghten, Richard A; Dooley, Colette T; Appel, Jon R

2006-05-26

The use of combinatorial libraries for the identification of novel opiate and related ligands in opioid receptor assays is reviewed. Case studies involving opioid assays used to demonstrate the viability of combinatorial libraries are described. The identification of new opioid peptides composed of L-amino acids, D-amino acids, or L-, D-, and unnatural amino acids is reviewed. New opioid compounds have also been identified from peptidomimetic libraries, such as peptoids and alkylated dipeptides, and those identified from acyclic (eg, polyamine, urea) and heterocyclic (eg, bicyclic guanidine) libraries are reviewed.

Transcript and proteomic analysis of developing white lupin (Lupinus albus L.) roots

PubMed Central

Tian, Li; Peel, Gregory J; Lei, Zhentian; Aziz, Naveed; Dai, Xinbin; He, Ji; Watson, Bonnie; Zhao, Patrick X; Sumner, Lloyd W; Dixon, Richard A

2009-01-01

Background White lupin (Lupinus albus L.) roots efficiently take up and accumulate (heavy) metals, adapt to phosphate deficiency by forming cluster roots, and secrete antimicrobial prenylated isoflavones during development. Genomic and proteomic approaches were applied to identify candidate genes and proteins involved in antimicrobial defense and (heavy) metal uptake and translocation. Results A cDNA library was constructed from roots of white lupin seedlings. Eight thousand clones were randomly sequenced and assembled into 2,455 unigenes, which were annotated based on homologous matches in the NCBInr protein database. A reference map of developing white lupin root proteins was established through 2-D gel electrophoresis and peptide mass fingerprinting. High quality peptide mass spectra were obtained for 170 proteins. Microsomal membrane proteins were separated by 1-D gel electrophoresis and identified by LC-MS/MS. A total of 74 proteins were putatively identified by the peptide mass fingerprinting and the LC-MS/MS methods. Genomic and proteomic analyses identified candidate genes and proteins encoding metal binding and/or transport proteins, transcription factors, ABC transporters and phenylpropanoid biosynthetic enzymes. Conclusion The combined EST and protein datasets will facilitate the understanding of white lupin's response to biotic and abiotic stresses and its utility for phytoremediation. The root ESTs provided 82 perfect simple sequence repeat (SSR) markers with potential utility in breeding white lupin for enhanced agronomic traits. PMID:19123941

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope.

PubMed

Navari, Mohsen; Zare, Mehrak; Javanmardi, Masoud; Asadi-Ghalehni, Majid; Modjtahedi, Helmout; Rasaee, Mohammad Javed

2014-10-01

One of the proposed approaches in cancer therapy is to induce and direct the patient's own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.

Hydroxyapatite-binding peptides for bone growth and inhibition

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

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.

Selection of peptides interfering with protein-protein interaction.

PubMed

Gaida, Annette; Hagemann, Urs B; Mattay, Dinah; Räuber, Christina; Müller, Kristian M; Arndt, Katja M

2009-01-01

Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing suitable selection techniques and ending with purification for further down-stream characterization. Such generated peptides will function as versatile tools to interfere with the natural function of their targets thereby illuminating their down-stream signaling and, in general, promoting understanding of factors leading to specificity and stability in protein-protein interactions. Furthermore, peptides interfering with medically relevant proteins might become important diagnostics and therapeutics.

Comparative characterization of random-sequence proteins consisting of 5, 12, and 20 kinds of amino acids

PubMed Central

Tanaka, Junko; Doi, Nobuhide; Takashima, Hideaki; Yanagawa, Hiroshi

2010-01-01

Screening of functional proteins from a random-sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random-sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random-sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random-sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279–284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120-amino acid, random-sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random-sequence proteins arbitrarily chosen from these libraries. We found that random-sequence proteins constructed with the 12-member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20-member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids. PMID:20162614

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.

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.

Heterodera glycines cysts contain an extensive array of endoproteases as well as inhibitors of proteases in H. glycines and Meloidogyne incognita infective juvenile stages

USDA-ARS?s Scientific Manuscript database

Heterodera glycines cysts contain proteases, and inhibitors of protease activities in various nematode species. In this investigation, proteases in H. glycines cysts were identified using a commercially available FRET-peptide library comprising 512 peptide pools qualified to detect up to 4 endoprot...

MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration

PubMed Central

2012-01-01

Naturally occurring native peptides provide important information about physiological states of an organism and its changes in disease conditions but protocols and methods for assessing their abundance are not well-developed. In this paper, we describe a simple procedure for the quantification of non-tryptic peptides in body fluids. The workflow includes an enrichment step followed by two-dimensional fractionation of native peptides and MS/MS data management facilitating the design and validation of LC- MRM MS assays. The added value of the workflow is demonstrated in the development of a triplex LC-MRM MS assay used for quantification of peptides potentially associated with the progression of liver disease to hepatocellular carcinoma. PMID:22304756

Mapping a disordered portion of the Brz2001-binding site on a plant monooxygenase, DWARF4, using a quartz-crystal microbalance biosensor-based T7 phage display.

PubMed

Takakusagi, Yoichi; Manita, Daisuke; Kusayanagi, Tomoe; Izaguirre-Carbonell, Jesus; Takakusagi, Kaori; Kuramochi, Kouji; Iwabata, Kazuki; Kanai, Yoshihiro; Sakaguchi, Kengo; Sugawara, Fumio

2013-04-01

In small-molecule/protein interaction studies, technical difficulties such as low solubility of small molecules or low abundance of protein samples often restrict the progress of research. Here, we describe a quartz-crystal microbalance (QCM) biosensor-based T7 phage display in combination use with a receptor-ligand contacts (RELIC) bioinformatics server for application in a plant Brz2001/DWARF4 system. Brz2001 is a brassinosteroid biosynthesis inhibitor in the less-soluble triazole series of compounds that targets DWARF4, a cytochrome P450 (Cyp450) monooxygenase containing heme and iron. Using a Brz2001 derivative that has higher solubility in 70% EtOH and forms a self-assembled monolayer on gold electrode, we selected 34 Brz2001-recognizing peptides from a 15-mer T7 phage-displayed random peptide library using a total of four sets of one-cycle biopanning. The RELIC/MOTIF program revealed continuous and discontinuous short motifs conserved within the 34 Brz2001-selected 15-mer peptide sequences, indicating the increase of information content for Brz2001 recognition. Furthermore, an analysis of similarity between the 34 peptides and the amino-acid sequence of DWARF4 using the RELIC/MATCH program generated a similarity plot and a cluster diagram of the amino-acid sequence. Both of these data highlighted an internally located disordered portion of a catalytic site on DWARF4, indicating that this portion is essential for Brz2001 recognition. A similar trend was also noted by an analysis using another 26 Brz2001-selected peptides, and not observed using the 27 gold electrode-recognizing control peptides, demonstrating the reproducibility and specificity of this method. Thus, this affinity-based strategy enables high-throughput detection of the small-molecule-recognizing portion on the target protein, which overcomes technical difficulties such as sample solubility or preparation that occur when conventional methods are used.

An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas' disease

PubMed Central

Assumpção, Teresa C. F.; Francischetti, Ivo M. B.; Andersen, John F.; Schwarz, Alexandra; Santana, Jaime M.; Ribeiro, José M. C.

2008-01-01

Triatoma infestans is a hemiptera, vector of Chagas’ disease, that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects’ salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anti-clotting, anti-platelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Also, salivary proteins were submitted to two dimentional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1,534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results. PMID:18207082

A novel spectral library workflow to enhance protein identifications.

PubMed

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

2013-04-09

The innovations in mass spectrometry-based investigations in proteome biology enable systematic characterization of molecular details in pathophysiological phenotypes. However, the process of delineating large-scale raw proteomic datasets into a biological context requires high-throughput data acquisition and processing. A spectral library search engine makes use of previously annotated experimental spectra as references for subsequent spectral analyses. This workflow delivers many advantages, including elevated analytical efficiency and specificity as well as reduced demands in computational capacity. In this study, we created a spectral matching engine to address challenges commonly associated with a library search workflow. Particularly, an improved sliding dot product algorithm, that is robust to systematic drifts of mass measurement in spectra, is introduced. Furthermore, a noise management protocol distinguishes spectra correlation attributed from noise and peptide fragments. It enables elevated separation between target spectral matches and false matches, thereby suppressing the possibility of propagating inaccurate peptide annotations from library spectra to query spectra. Moreover, preservation of original spectra also accommodates user contributions to further enhance the quality of the library. Collectively, this search engine supports reproducible data analyses using curated references, thereby broadening the accessibility of proteomics resources to biomedical investigators. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules.

PubMed

Binkowski, Brock F; Miller, Russell A; Belshaw, Peter J

2005-07-01

We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.

Cloning of precursors for two MIH/VIH-related peptides in the prawn, Macrobrachium rosenbergii.

PubMed

Yang, W J; Rao, K R

2001-11-30

Two cDNA clones (634 and 1366 bp) encoding MIH/VIH (molt-inhibiting hormone/vitellogenesis-inhibiting hormone)-related peptides were isolated and sequenced from a Macrobrachium rosenbergii eyestalk ganglia cDNA library. The clones contain a 360 and 339 bp open-reading frame, and their conceptually translated peptides consist of a 41 and 34 amino acid signal peptide, respectively, and a 78 amino acid residue mature peptide hormone. The amino acid sequences of the peptides exhibit higher identities with other known MIHs and VIH (44-69%) than with CHHs (28-33%). This is the first report describing the cloning and sequencing of two MIH/VIH-related peptides in a single crustacean species. Transcription of these mRNAs was detected in the eyestalk ganglia, but not in the thoracic ganglia, hepatopancreas, gut, gill, heart, or muscle.

Intravenous phage display identifies peptide sequences that target the burn-injured intestine.

PubMed

Costantini, Todd W; Eliceiri, Brian P; Putnam, James G; Bansal, Vishal; Baird, Andrew; Coimbra, Raul

2012-11-01

The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 10(12) phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1×10(12) copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4-1: SGHQLLLNKMP, 4-5: ILANDLTAPGPR, 4-11: SFKPSGLPAQSL). Sequence 4-5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9×10(5)vs. 3.1×10(4) particles per mg tissue). Sequences 4-1 and 4-11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4-11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Characterization of diverse internal binding specificities of PDZ domains by yeast two-hybrid screening of a special peptide library.

PubMed

Mu, Yi; Cai, Pengfei; Hu, Siqi; Ma, Sucan; Gao, Youhe

2014-01-01

Protein-protein interactions (PPIs) are essential events to play important roles in a series of biological processes. There are probably more ways of PPIs than we currently realized. Structural and functional investigations of weak PPIs have lagged behind those of strong PPIs due to technical difficulties. Weak PPIs are often short-lived, which may result in more dynamic signals with important biological roles within and/or between cells. For example, the characteristics of PSD-95/Dlg/ZO-1 (PDZ) domain binding to internal sequences, which are primarily weak interactions, have not yet been systematically explored. In the present study, we constructed a nearly random octapeptide yeast two-hybrid library. A total of 24 PDZ domains were used as baits for screening the library. Fourteen of these domains were able to bind internal PDZ-domain binding motifs (PBMs), and PBMs screened for nine PDZ domains exhibited strong preferences. Among 11 PDZ domains that have not been reported their internal PBM binding ability, six were confirmed to bind internal PBMs. The first PDZ domain of LNX2, which has not been reported to bind C-terminal PBMs, was found to bind internal PBMs. These results suggest that the internal PBMs binding ability of PDZ domains may have been underestimated. The data provided diverse internal binding properties for several PDZ domains that may help identify their novel binding partners.

Identification of three novel B-cell epitopes of VMH protein from Vibrio mimicus by screening a phage display peptide library.

PubMed

Xiao, Ning; Cao, Ji; Zhou, Hao; Ding, Shu-Quan; Kong, Ling-Yan; Li, Jin-Nian

2016-12-01

Vibrio mimicus is the causative agent of ascites disease in fish. The heat-labile hemolytic toxin designated VMH is an immunoprotective antigen of V. mimicus. However, its epitopes have not been well characterized. Here, a commercially available phage displayed 12-mer peptide library was used to screen epitopes of VMH protein using polyclonal rabbit anti-rVMH protein antibodies, and then five positive phage clones were identified by sandwich and competitive ELISA. Sequences analysis showed that the motif of DPTLL displayed on phage clone 15 and the consensus motif of SLDDDST displayed on the clone 4/11 corresponded to the residues 134-138 and 238-244 of VMH protein, respectively, and the synthetic motif peptides could also be recognized by anti-rVMH-HD antibody in peptide-ELISA. Thus, both motifs DPTLL and SLDDDST were identified as minimal linear B-cell epitopes of VMH protein. Although no similarity was found between VMH protein and the consensus motif of ADGLVPR displayed on the clone 2/6, the synthetic peptide ADGLVPR could absorb anti-rVMH-HD antibody and inhibit the antibody binding to rVMH protein in enhanced chemoluminescence Western blotting, whereas irrelevant control peptide did not affect the antibody binding with rVMH. These results revealed that the peptide ADGLVPR was a mimotope of VMH protein. Taken together, three novel B-cell epitopes of VMH protein were identified, which provide a foundation for developing epitope-based vaccine against V. mimicus infection in fish. Copyright © 2016 Elsevier B.V. All rights reserved.

Peptides Derived from a Phage Display Library Inhibit Adhesion and Protect the Host against Infection by Paracoccidioides brasiliensis and Paracoccidioides lutzii

PubMed Central

de Oliveira, Haroldo C.; Michaloski, Jussara S.; da Silva, Julhiany F.; Scorzoni, Liliana; de Paula e Silva, Ana C. A.; Marcos, Caroline M.; Assato, Patrícia A.; Yamazaki, Daniella S.; Fusco-Almeida, Ana M.; Giordano, Ricardo J.; Mendes-Giannini, Maria J. S.

2016-01-01

Paracoccidioides brasiliensis and Paracoccidioides lutzii are dimorphic fungi and are the etiological agents of paracoccidioidomycosis (PCM). Adhesion is one of the most important steps in infections with Paracoccidioides and is responsible for the differences in the virulence of isolates of these fungi. Because of the importance of adhesion to the establishment of an infection, this study focused on the preliminary development of a new therapeutic strategy to inhibit adhesion by Paracoccidioides, thus inhibiting infection and preventing the disease. We used two phage display libraries to select peptides that strongly bind to the Paracoccidioides cell wall to inhibit adhesion to host cells and extracellular matrix (ECM) components (laminin, fibronectin, and type I and type IV collagen). This approach allowed us to identify four peptides that inhibited up to 64% of the adhesion of Paracoccidioides to pneumocytes in vitro and inhibited the adhesion to the ECM components by up to 57%. Encouraged by these results, we evaluated the ability of these peptides to protect Galleria mellonella from Paracoccidioides infection by treating G. mellonella larvae with the different peptides prior to infection with Paracoccidioides and observing larval survival. The results show that all of the peptides tested increased the survival of the larvae infected with P. brasiliensis by up to 64% and by up to 60% in those infected with P. lutzii. These data may open new horizons for therapeutic strategies to prevent PCM, and anti-adhesion therapy could be an important strategy. PMID:28066254

Selection of peptides targeting helix 31 of bacterial 16S ribosomal RNA by screening M13 phage-display libraries.

PubMed

Lamichhane, Tek N; Abeydeera, N Dinuka; Duc, Anne-Cécile E; Cunningham, Philip R; Chow, Christine S

2011-01-28

Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational changes during translation. Structural changes in ribosomal RNA can be facilitated by the presence of modified nucleotides. Helix 31 of bacterial 16S ribosomal RNA harbors two modified nucleotides, m²G966 and m⁵C967, that are highly conserved among bacteria, though the degree and nature of the modifications in this region are different in eukaryotes. Contacts between helix 31 and the P-site tRNA, initiation factors, and ribosomal proteins highlight the importance of this region in translation. In this work, a heptapeptide M13 phage-display library was screened for ligands that target the wild-type, naturally modified bacterial helix 31. Several peptides, including TYLPWPA, CVRPFAL, TLWDLIP, FVRPFPL, ATPLWLK, and DIRTQRE, were found to be prevalent after several rounds of screening. Several of the peptides exhibited moderate affinity (in the high nM to low µM range) to modified helix 31 in biophysical assays, including surface plasmon resonance (SPR), and were also shown to bind 30S ribosomal subunits. These peptides also inhibited protein synthesis in cell-free translation assays.

A solid-phase combinatorial approach for indoloquinolizidine-peptides with high affinity at D(1) and D(2) dopamine receptors.

PubMed

Molero, Anabel; Vendrell, Marc; Bonaventura, Jordi; Zachmann, Julian; López, Laura; Pardo, Leonardo; Lluis, Carme; Cortés, Antoni; Albericio, Fernando; Casadó, Vicent; Royo, Miriam

2015-06-05

Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D1R and D2R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D2R, a receptor that is crucial to re-establish activity on dopamine-depleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D2R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D1 and D2 dopamine receptors. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Proteomic analysis of sweet algerian apricot kernels (Prunus armeniaca L.) by combinatorial peptide ligand libraries and LC-MS/MS.

PubMed

Ghorab, Hamida; Lammi, Carmen; Arnoldi, Anna; Kabouche, Zahia; Aiello, Gilda

2018-01-15

An investigation on the proteome of the sweet kernel of apricot, based on equalisation with combinatorial peptide ligand libraries (CPLLs), SDS-PAGE, nLC-ESI-MS/MS, and database search, permitted identifying 175 proteins. Gene ontology analysis indicated that their main molecular functions are in nucleotide binding (20.9%), hydrolase activities (10.6%), kinase activities (7%), and catalytic activity (5.6%). A protein-protein association network analysis using STRING software permitted to build an interactomic map of all detected proteins, characterised by 34 interactions. In order to forecast the potential health benefits deriving from the consumption of these proteins, the two most abundant, i.e. Prunin 1 and 2, were enzymatically digested in silico predicting 10 and 14 peptides, respectively. Searching their sequences in the database BIOPEP, it was possible to suggest a variety of bioactivities, including dipeptidyl peptidase-IV (DPP-IV) and angiotensin converting enzyme I (ACE) inhibition, glucose uptake stimulation and antioxidant properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

MacroEvoLution: A New Method for the Rapid Generation of Novel Scaffold-Diverse Macrocyclic Libraries.

PubMed

Saupe, Jörn; Kunz, Oliver; Haustedt, Lars Ole; Jakupovic, Sven; Mang, Christian

2017-09-04

Macrocycles are a structural class bearing great promise for future challenges in medicinal chemistry. Nevertheless, there are few flexible approaches for the rapid generation of structurally diverse macrocyclic compound collections. Here, an efficient method for the generation of novel macrocyclic peptide-based scaffolds is reported. The process, named here as "MacroEvoLution", is based on a cyclization screening approach that gives reliable access to novel macrocyclic architectures. Classification of building blocks into specific pools ensures that scaffolds with orthogonally addressable functionalities are generated, which can easily be used for the generation of structurally diverse compound libraries. The method grants rapid access to novel scaffolds with scalable synthesis (multi gram scale) and the introduction of further diversity at a late stage. Despite being developed for peptidic systems, the approach can easily be extended for the synthesis of systems with a decreased peptidic character. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Direct Identification of On-Bead Peptides Using Surface-Enhanced Raman Spectroscopic Barcoding System for High-Throughput Bioanalysis

PubMed Central

Kang, Homan; Jeong, Sinyoung; Koh, Yul; Geun Cha, Myeong; Yang, Jin-Kyoung; Kyeong, San; Kim, Jaehi; Kwak, Seon-Yeong; Chang, Hye-Jin; Lee, Hyunmi; Jeong, Cheolhwan; Kim, Jong-Ho; Jun, Bong-Hyun; Kim, Yong-Kweon; Hong Jeong, Dae; Lee, Yoon-Sik

2015-01-01

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery. PMID:26017924

Direct identification of on-bead peptides using surface-enhanced Raman spectroscopic barcoding system for high-throughput bioanalysis.

PubMed

Kang, Homan; Jeong, Sinyoung; Koh, Yul; Geun Cha, Myeong; Yang, Jin-Kyoung; Kyeong, San; Kim, Jaehi; Kwak, Seon-Yeong; Chang, Hye-Jin; Lee, Hyunmi; Jeong, Cheolhwan; Kim, Jong-Ho; Jun, Bong-Hyun; Kim, Yong-Kweon; Hong Jeong, Dae; Lee, Yoon-Sik

2015-05-28

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery.

Spectral Archives: Extending Spectral Libraries to Analyze both Identified and Unidentified Spectra

PubMed Central

Frank, Ari M.; Monroe, Matthew E.; Shah, Anuj R.; Carver, Jeremy J.; Bandeira, Nuno F.; Moore, Ronald J.; Anderson, Gordon A.; Smith, Richard D.; Pevzner, Pavel A.

2011-01-01

MS/MS experiments generate multiple, nearly identical spectra of the same peptide in various laboratories, but proteomics researchers typically do not leverage the unidentified spectra produced in other labs to decode spectra generated in their own labs. We propose a spectral archives approach that clusters MS/MS datasets, representing similar spectra by a single consensus spectrum. Spectral archives extend spectral libraries by analyzing both identified and unidentified spectra in the same way and maintaining information about spectra of peptides shared across species and conditions. Thus archives offer both traditional library spectrum similarity-based search capabilities along with novel ways to analyze the data. By developing a clustering tool, MS-Cluster, we generated a spectral archive from ~1.18 billion spectra that greatly exceeds the size of existing spectral repositories. We advocate that publicly available data should be organized into spectral archives, rather than be analyzed as disparate datasets, as is mostly the case today. PMID:21572408

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

2011-05-06

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.

Isolation of a peptide from Ph.D.-C7C phage display library for detection of Cry1Ab.

PubMed

Wang, Yun; Wang, Qian; Wu, Ai-Hua; Hao, Zhen-Ping; Liu, Xian-Jin

2017-12-15

Traditional ELISA methods of using animal immunity yield antibodies for detection Cry toxin. Not only is this incredibly harmful to the animals, but is also time-intensive. Here we developed a simple method to yield the recognition element. Using a critical selection strategy and immunoassay we confirmed a clone from the Ph.D-C7C phage library, which has displayed the most interesting Cry1Ab-binding characteristics examined in this study (Fig. 1). The current study indicates that isolating peptide is an alternative method for the preparation of a recognition element, and that the developed assay is a potentially useful tool for detecting Cry1Ab. Copyright © 2017. Published by Elsevier Inc.

Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection.

PubMed

Tikunova, N V; Morozova, V V

2009-10-01

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

NASA Astrophysics Data System (ADS)

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-03-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

PubMed Central

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-01-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071–40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands. PMID:24670678

Single-carbon discrimination by selected peptides for individual detection of volatile organic compounds

NASA Astrophysics Data System (ADS)

Ju, Soomi; Lee, Ki-Young; Min, Sun-Joon; Yoo, Yong Kyoung; Hwang, Kyo Seon; Kim, Sang Kyung; Yi, Hyunjung

2015-03-01

Although volatile organic compounds (VOCs) are becoming increasingly recognized as harmful agents and potential biomarkers, selective detection of the organic targets remains a tremendous challenge. Among the materials being investigated for target recognition, peptides are attractive candidates because of their chemical robustness, divergence, and their homology to natural olfactory receptors. Using a combinatorial peptide library and either a graphitic surface or phenyl-terminated self-assembled monolayer as relevant target surfaces, we successfully selected three interesting peptides that differentiate a single carbon deviation among benzene and its analogues. The heterogeneity of the designed target surfaces provided peptides with varying affinity toward targeted molecules and generated a set of selective peptides that complemented each other. Microcantilever sensors conjugated with each peptide quantitated benzene, toluene and xylene to sub-ppm levels in real time. The selection of specific receptors for a group of volatile molecules will provide a strong foundation for general approach to individually monitoring VOCs.

Quantitating T cell cross-reactivity for unrelated peptide antigens.

PubMed

Ishizuka, Jeffrey; Grebe, Kristie; Shenderov, Eugene; Peters, Bjoern; Chen, Qiongyu; Peng, Yanchun; Wang, Lili; Dong, Tao; Pasquetto, Valerie; Oseroff, Carla; Sidney, John; Hickman, Heather; Cerundolo, Vincenzo; Sette, Alessandro; Bennink, Jack R; McMichael, Andrew; Yewdell, Jonathan W

2009-10-01

Quantitating the frequency of T cell cross-reactivity to unrelated peptides is essential to understanding T cell responses in infectious and autoimmune diseases. Here we used 15 mouse or human CD8+ T cell clones (11 antiviral, 4 anti-self) in conjunction with a large library of defined synthetic peptides to examine nearly 30,000 TCR-peptide MHC class I interactions for cross-reactions. We identified a single cross-reaction consisting of an anti-self TCR recognizing a poxvirus peptide at relatively low sensitivity. We failed to identify any cross-reactions between the synthetic peptides in the panel and polyclonal CD8+ T cells raised to viral or alloantigens. These findings provide the best estimate to date of the frequency of T cell cross-reactivity to unrelated peptides ( approximately 1/30,000), explaining why cross-reactions between unrelated pathogens are infrequently encountered and providing a critical parameter for understanding the scope of self-tolerance.

Quantitating T Cell Cross-Reactivity for Unrelated Peptide Antigens1

PubMed Central

Ishizuka, Jeffrey; Grebe, Kristie; Shenderov, Eugene; Peters, Bjoern; Chen, Qiongyu; Peng, YanChun; Wang, Lili; Dong, Tao; Pasquetto, Valerie; Osroff, Carla; Sidney, John; Hickman, Heather; Cerundolo, Vincenzo; Sette, Alessandro; Bennink, Jack R.; McMchael, Andrew; Yewdell, Jonathan W.

2009-01-01

Quantitating the frequency of T cell cross-reactivity to unrelated peptides is essential to understanding T cell responses in infectious and autoimmune diseases. Here we used 15 mouse or human CD8+ T cell clones (11 antiviral, 4 anti-self) in conjunction with a large library of defined synthetic peptides to examine nearly 30,000 TCR-peptide MHC class I interactions for cross-reactions. We identified a single cross-reaction consisting of an anti-self TCR recognizing a poxvirus peptide at relatively low sensitivity. We failed to identify any cross-reactions between the synthetic peptides in the panel and polyclonal CD8+ T cells raised to viral or alloantigens. These findings provide the best estimate to date of the frequency of T cell cross-reactivity to unrelated peptides (∼1/30,000), explaining why cross-reactions between unrelated pathogens are infrequently encountered and providing a critical parameter for understanding the scope of self-tolerance. PMID:19734234

Correlation between y-type ions observed in ion trap and triple quadrupole mass spectrometers.

PubMed

Sherwood, Carly A; Eastham, Ashley; Lee, Lik Wee; Risler, Jenni; Vitek, Olga; Martin, Daniel B

2009-09-01

Multiple reaction monitoring mass spectrometry (MRM-MS) is a technique for high-sensitivity targeted analysis. In proteomics, MRM-MS can be used to monitor and quantify a peptide based on the production of expected fragment peaks from the selected peptide precursor ion. The choice of which fragment ions to monitor in order to achieve maximum sensitivity in MRM-MS can potentially be guided by existing MS/MS spectra. However, because the majority of discovery experiments are performed on ion trap platforms, there is concern in the field regarding the generalizability of these spectra to MRM-MS on a triple quadrupole instrument. In light of this concern, many operators perform an optimization step to determine the most intense fragments for a target peptide on a triple quadrupole mass spectrometer. We have addressed this issue by targeting, on a triple quadrupole, the top six y-ion peaks from ion trap-derived consensus library spectra for 258 doubly charged peptides from three different sample sets and quantifying the observed elution curves. This analysis revealed a strong correlation between the y-ion peak rank order and relative intensity across platforms. This suggests that y-type ions obtained from ion trap-based library spectra are well-suited for generating MRM-MS assays for triple quadrupoles and that optimization is not required for each target peptide.

Toward the characterization of biological toxins using field-based FT-IR spectroscopic instrumentation

NASA Astrophysics Data System (ADS)

Schiering, David W.; Walton, Robert B.; Brown, Christopher W.; Norman, Mark L.; Brewer, Joseph; Scott, James

2004-12-01

IR spectroscopy is a broadly applicable technique for the identification of covalent materials. Recent advances in instrumentation have made Fourier Transform infrared (FT-IR) spectroscopy available for field characterization of suspect materials. Presently, this instrumentation is broadly deployed and used for the identification of potential chemical hazards. This discussion concerns work towards expanding the analytical utility of field-based FT-IR spectrometry in the characterization of biological threats. Two classes of materials were studied: biologically produced chemical toxins which were non-peptide in nature and peptide toxin. The IR spectroscopic identification of aflatoxin-B1, trichothecene T2 mycotoxin, and strychnine was evaluated using the approach of spectral searching against large libraries of materials. For pure components, the IR method discriminated the above toxins at better than the 99% confidence level. The ability to identify non-peptide toxins in mixtures was also evaluated using a "spectral stripping" search approach. For the mixtures evaluated, this method was able to identify the mixture components from ca. 32K spectral library entries. Castor bean extract containing ricin was used as a representative peptide toxin. Due to similarity in protein spectra, a SIMCA pattern recognition methodology was evaluated for classifying peptide toxins. In addition to castor bean extract the method was validated using bovine serum albumin and myoglobin as simulants. The SIMCA approach was successful in correctly classifying these samples at the 95% confidence level.

Can Biomarkers Help Target Maturity-Onset Diabetes of the Young Genetic Testing in Antibody-Negative Diabetes?

PubMed

Majidi, Shideh; Fouts, Alexandra; Pyle, Laura; Chambers, Christina; Armstrong, Taylor; Wang, Zhenyuan; Batish, Sat Dev; Klingensmith, Georgeanna; Steck, Andrea K

2018-02-01

Maturity-onset diabetes of the young (MODY) is an antibody-negative, autosomal dominant form of diabetes. With the increasing prevalence of diabetes and the expense of MODY testing, markers to identify those who need further genetic testing would be beneficial. We investigated whether HLA genotypes, random C-peptide, and/or high-sensitivity C-reactive protein (hsCRP) levels could be helpful biomarkers for identifying MODY in antibody-negative diabetes. Subjects (N = 97) with diabetes onset ≤age 25, measurable C-peptide (≥0.1 ng/mL), and negative for all four diabetes autoantibodies were enrolled at a large academic center and tested for MODY 1-5 through Athena Diagnostics. A total of 22 subjects had a positive or very likely pathogenic mutation for MODY. Random C-peptide levels were significantly different between MODY-positive and MODY-negative subjects (0.16 nmol/L vs. 0.02 nmol/L; P = 0.02). After adjusting for age and diabetes duration, hsCRP levels were significantly lower in MODY-positive subjects (0.37 mg/L vs. 0.87 mg/L; P = 0.02). Random C-peptide level ≥0.15 nmol/L obtained at ≥6 months after diagnosis had 83% sensitivity for diagnosis of MODY with a negative predictive value of 96%. Receiver operating characteristic curves showed that area under the curve for random C-peptide (0.75) was significantly better than hsCRP (0.54), high-risk HLA DR3/4-DQB1*0302 (0.59), and high-risk HLA/random C-peptide combined (0.54; P = 0.03). Random C-peptide obtained at ≥6 months after diagnosis can be a useful biomarker to identify antibody-negative individuals who need further genetic testing for MODY, whereas hsCRP and HLA do not appear to improve this antibody/C-peptide-based approach.

[Progress in the spectral library based protein identification strategy].

PubMed

Yu, Derui; Ma, Jie; Xie, Zengyan; Bai, Mingze; Zhu, Yunping; Shu, Kunxian

2018-04-25

Exponential growth of the mass spectrometry (MS) data is exhibited when the mass spectrometry-based proteomics has been developing rapidly. It is a great challenge to develop some quick, accurate and repeatable methods to identify peptides and proteins. Nowadays, the spectral library searching has become a mature strategy for tandem mass spectra based proteins identification in proteomics, which searches the experiment spectra against a collection of confidently identified MS/MS spectra that have been observed previously, and fully utilizes the abundance in the spectrum, peaks from non-canonical fragment ions, and other features. This review provides an overview of the implement of spectral library search strategy, and two key steps, spectral library construction and spectral library searching comprehensively, and discusses the progress and challenge of the library search strategy.

Selection, Design and Applications of Solid Binding Peptides for Controlled Biomineralization

NASA Astrophysics Data System (ADS)

Gungormus, Mustafa

The regulated formation of inorganic minerals in living systems is called biomineralization. The mineralization processes of biogenic minerals are strictly regulated by a specific set of biomolecules, typically by proteins. Biologically formed calcium phosphates (hydroxyapatite (HA) in particular) are almost always nano-dimensional and nano-crystalline and are formed in vivo under mild conditions. The importance of understanding the molecular mechanisms underlying biological mineralization is underscored by the diseases associated with ectopic calcification or defects of skeletal mineralization. Proteins that mediate the mineralization process offer us a template for understanding how to potentially enhance the treatment of calcified tissue defects. However, the complexity and the number of proteins involved even in a rather simple biomineralization process can be daunting. The goal of this research was to identify short peptides through peptide-display libraries and demonstrate the utility of these peptides in better understanding the molecular mechanisms underlying biomineralization and as potential therapeutic agents in repairing calcified tissue defects. Using the phage-peptide display library, we have identified peptides that have affinity to HA and can regulate the formation of HA crystals. Utilizing the sequence knowledge obtained, we have identified putative functional domains within amelogenin; a naturally existing mineralization related protein, which, in turn, may provide a better understanding of the formation of dental enamel. These peptides also have the potential to be used to repair calcified tissue defects. To investigate this, we have developed and tested in vitro a hydrogel scaffold with pre-determined functionalities. We have also demonstrated the feasibility of obtaining significant re-mineralization of incipient dental root lesions ex vivo in a relatively short time using the mineralization-directing peptides. The approaches developed through this research may offer clinically acceptable novel procedures to treat calcified tissue defects because the developed peptide is safe to use and promotes the mineralization in a short period of time offered by none of the existing treatment options.

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

PubMed

Hoopmann, Michael R; Moritz, Robert L

2013-02-01

Peptide-based proteomic data sets are ever increasing in size and complexity. These data sets provide computational challenges when attempting to quickly analyze spectra and obtain correct protein identifications. Database search and de novo algorithms must consider high-resolution MS/MS spectra and alternative fragmentation methods. Protein inference is a tricky problem when analyzing large data sets of degenerate peptide identifications. Combining multiple algorithms for improved peptide identification puts significant strain on computational systems when investigating large data sets. This review highlights some of the recent developments in peptide and protein identification algorithms for analyzing shotgun mass spectrometry data when encountering the aforementioned hurdles. Also explored are the roles that analytical pipelines, public spectral libraries, and cloud computing play in the evolution of peptide-based proteomics. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

reSpect: Software for Identification of High and Low Abundance Ion Species in Chimeric Tandem Mass Spectra

PubMed Central

Shteynberg, David; Mendoza, Luis; Hoopmann, Michael R.; Sun, Zhi; Schmidt, Frank; Deutsch, Eric W.; Moritz, Robert L.

2016-01-01

Most shotgun proteomics data analysis workflows are based on the assumption that each fragment ion spectrum is explained by a single species of peptide ion isolated by the mass spectrometer; however, in reality mass spectrometers often isolate more than one peptide ion within the window of isolation that contributes to additional peptide fragment peaks in many spectra. We present a new tool called reSpect, implemented in the Trans-Proteomic Pipeline (TPP), that enables an iterative workflow whereby fragment ion peaks explained by a peptide ion identified in one round of sequence searching or spectral library search are attenuated based on the confidence of the identification, and then the altered spectrum is subjected to further rounds of searching. The reSpect tool is not implemented as a search engine, but rather as a post search engine processing step where only fragment ion intensities are altered. This enables the application of any search engine combination in the following iterations. Thus, reSpect is compatible with all other protein sequence database search engines as well as peptide spectral library search engines that are supported by the TPP. We show that while some datasets are highly amenable to chimeric spectrum identification and lead to additional peptide identification boosts of over 30% with as many as four different peptide ions identified per spectrum, datasets with narrow precursor ion selection only benefit from such processing at the level of a few percent. We demonstrate a technique that facilitates the determination of the degree to which a dataset would benefit from chimeric spectrum analysis. The reSpect tool is free and open source, provided within the TPP and available at the TPP website. PMID:26419769

reSpect: software for identification of high and low abundance ion species in chimeric tandem mass spectra.

PubMed

Shteynberg, David; Mendoza, Luis; Hoopmann, Michael R; Sun, Zhi; Schmidt, Frank; Deutsch, Eric W; Moritz, Robert L

2015-11-01

Most shotgun proteomics data analysis workflows are based on the assumption that each fragment ion spectrum is explained by a single species of peptide ion isolated by the mass spectrometer; however, in reality mass spectrometers often isolate more than one peptide ion within the window of isolation that contribute to additional peptide fragment peaks in many spectra. We present a new tool called reSpect, implemented in the Trans-Proteomic Pipeline (TPP), which enables an iterative workflow whereby fragment ion peaks explained by a peptide ion identified in one round of sequence searching or spectral library search are attenuated based on the confidence of the identification, and then the altered spectrum is subjected to further rounds of searching. The reSpect tool is not implemented as a search engine, but rather as a post-search engine processing step where only fragment ion intensities are altered. This enables the application of any search engine combination in the iterations that follow. Thus, reSpect is compatible with all other protein sequence database search engines as well as peptide spectral library search engines that are supported by the TPP. We show that while some datasets are highly amenable to chimeric spectrum identification and lead to additional peptide identification boosts of over 30% with as many as four different peptide ions identified per spectrum, datasets with narrow precursor ion selection only benefit from such processing at the level of a few percent. We demonstrate a technique that facilitates the determination of the degree to which a dataset would benefit from chimeric spectrum analysis. The reSpect tool is free and open source, provided within the TPP and available at the TPP website. Graphical Abstract ᅟ.

reSpect: Software for Identification of High and Low Abundance Ion Species in Chimeric Tandem Mass Spectra

NASA Astrophysics Data System (ADS)

Shteynberg, David; Mendoza, Luis; Hoopmann, Michael R.; Sun, Zhi; Schmidt, Frank; Deutsch, Eric W.; Moritz, Robert L.

2015-11-01

Most shotgun proteomics data analysis workflows are based on the assumption that each fragment ion spectrum is explained by a single species of peptide ion isolated by the mass spectrometer; however, in reality mass spectrometers often isolate more than one peptide ion within the window of isolation that contribute to additional peptide fragment peaks in many spectra. We present a new tool called reSpect, implemented in the Trans-Proteomic Pipeline (TPP), which enables an iterative workflow whereby fragment ion peaks explained by a peptide ion identified in one round of sequence searching or spectral library search are attenuated based on the confidence of the identification, and then the altered spectrum is subjected to further rounds of searching. The reSpect tool is not implemented as a search engine, but rather as a post-search engine processing step where only fragment ion intensities are altered. This enables the application of any search engine combination in the iterations that follow. Thus, reSpect is compatible with all other protein sequence database search engines as well as peptide spectral library search engines that are supported by the TPP. We show that while some datasets are highly amenable to chimeric spectrum identification and lead to additional peptide identification boosts of over 30% with as many as four different peptide ions identified per spectrum, datasets with narrow precursor ion selection only benefit from such processing at the level of a few percent. We demonstrate a technique that facilitates the determination of the degree to which a dataset would benefit from chimeric spectrum analysis. The reSpect tool is free and open source, provided within the TPP and available at the TPP website.

Discovery and application of peptides that bind to proteins and solid state inorganic materials

NASA Astrophysics Data System (ADS)

Stearns, Linda A.

A series of three projects was undertaken on the theme of peptide-based molecular recognition. In the first project, a messenger RNA (mRNA) display selection was carried out against the II-VI semiconductors zinc sulfide (ZnS), zinc selenide (ZnSe), and cadmium sulfide (CdS). Sequence analysis of 18-mer semiconductor-binding peptides (SBPs) following four rounds of selection indicated that the amino acid sequences were enriched in polar residues compared to the naive library, suggesting that hydrogen-bonding interactions are a dominant mode of interaction between the SBPs and their cognate inorganic surfaces. Select peptides were expressed as fusions of the green fluorescent protein (GFP) to visualize their recognition of semiconductor crystals. Interpretation of the results was complicated by a high fluorescence background that was observed with certain control GFP fusions. Additional experiments, including cross-specificity binding assays, are needed to characterize the peptides that were isolated in this selection. A second project described the practical application of a known inorganic-binding and nucleating peptide. Peptide A3, which was previously isolated by phage display, was chemically conjugated to a short DNA strand using the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). The resulting peptide-DNA conjugate was hybridized to ten complementary single-stranded capture probes extending outward from the surface of an origami DNA nanotube. A gold precursor solution was added to initiate nucleation and growth of gold nanoparticles at the site of the peptide. Transmission electron microscopy (TEM) was used to visualize the gold nanoparticle-decorated nanostructures. This approach holds immense promise for organizing compositionally-diverse materials at the nanoscale. In a third project, a novel non-iterative approach to mRNA display called covalent capture was demonstrated. Using human transferrin as a target protein, peptides with low-nanomolar affinity were isolated from a combinatorial library of one trillion distinct 12-mer peptide sequences by using UV light to covalently crosslink the peptides to a photoreactive arm that was displayed on the protein surface. The best peptide isolated from this screen exhibited a binding affinity constant (Kd) of 3 nM, which is equivalent to some of the best peptides isolated after many rounds of traditional bead-based selection. The approach itself is general and could be applied to many different types of problems in molecular biology.

Biomining with bacteriophage: selectivity of displayed peptides for naturally occurring sphalerite and chalcopyrite.

PubMed

Curtis, Susan B; Hewitt, Jeff; Macgillivray, Ross T A; Dunbar, W Scott

2009-02-01

During mineral processing, concentrates of sulfide minerals of economic interest are formed by froth flotation of fine ore particles. The method works well but recovery and selectivity can be poor for ores with complex mineralogy. There is considerable interest in methods that improve the selectivity of this process while avoiding the high costs of using flotation chemicals. Here we show the first application of phage biotechnology to the processing of economically important minerals in ore slurries. A random heptapeptide library was screened for peptide sequences that bind selectively to the minerals sphalerite (ZnS) and chalcopyrite (CuFeS2). After several rounds of enrichment, cloned phage containing the surface peptide loops KPLLMGS and QPKGPKQ bound specifically to sphalerite. Phage containing the peptide loop TPTTYKV bound to both sphalerite and chalcopyrite. By using an enzyme-linked immunosorbant assay (ELISA), the phage was characterized as strong binders compared to wild-type phage. Specificity of binding was confirmed by immunochemical visualization of phage bound to mineral particles but not to silica (a waste mineral) or pyrite. The current study focused primarily on the isolation of ZnS-specific phage that could be utilized in the separation of sphalerite from silica. At mining sites where sphalerite and chalcopyrite are not found together in natural ores, the separation of sphalerite from silica would be an appropriate enrichment step. At mining sites where sphalerite and chalcopyrite do occur together, more specific phage would be required. This bacteriophage has the potential to be used in a more selective method of mineral separation and to be the basis for advanced methods of mineral processing.

Differentiation of Streptococcus pneumoniae Conjunctivitis Outbreak Isolates by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry▿

PubMed Central

Williamson, Yulanda M.; Moura, Hercules; Woolfitt, Adrian R.; Pirkle, James L.; Barr, John R.; Carvalho, Maria Da Gloria; Ades, Edwin P.; Carlone, George M.; Sampson, Jacquelyn S.

2008-01-01

Streptococcus pneumoniae (pneumococcus [Pnc]) is a causative agent of many infectious diseases, including pneumonia, septicemia, otitis media, and conjunctivitis. There have been documented conjunctivitis outbreaks in which nontypeable (NT), nonencapsulated Pnc has been identified as the etiological agent. The use of mass spectrometry to comparatively and differentially analyze protein and peptide profiles of whole-cell microorganisms remains somewhat uncharted. In this report, we discuss a comparative proteomic analysis between NT S. pneumoniae conjunctivitis outbreak strains (cPnc) and other known typeable or NT pneumococcal and streptococcal isolates (including Pnc TIGR4 and R6, Streptococcus oralis, Streptococcus mitis, Streptococcus pseudopneumoniae, and Streptococcus pyogenes) and nonstreptococcal isolates (including Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus) as controls. cPnc cells and controls were grown to mid-log phase, harvested, and subsequently treated with a 10% trifluoroacetic acid-sinapinic acid matrix mixture. Protein and peptide fragments of the whole-cell bacterial isolate-matrix combinations ranging in size from 2 to 14 kDa were evaluated by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Additionally Random Forest analytical tools and dendrogramic representations (Genesis) suggested similarities and clustered the isolates into distinct clonal groups, respectively. Also, a peak list of protein and peptide masses was obtained and compared to a known Pnc protein mass library, in which a peptide common and unique to cPnc isolates was tentatively identified. Information gained from this study will lead to the identification and validation of proteins that are commonly and exclusively expressed in cPnc strains which could potentially be used as a biomarker in the rapid diagnosis of pneumococcal conjunctivitis. PMID:18708515

Conversion of scFv peptide-binding specificity for crystal chaperone development

PubMed Central

Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A.

2011-01-01

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 Å resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a ∼52 Å 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. PMID:21217145

Bimodal imprint chips for peptide screening: integration of high-throughput sequencing by MS and affinity analyses by surface plasmon resonance imaging.

PubMed

Wang, Weizhi; Li, Menglin; Wei, Zewen; Wang, Zihua; Bu, Xiangli; Lai, Wenjia; Yang, Shu; Gong, He; Zheng, Hui; Wang, Yuqiao; Liu, Ying; Li, Qin; Fang, Qiaojun; Hu, Zhiyuan

2014-04-15

Peptide probes and drugs have widespread applications in disease diagnostics and therapy. The demand for peptides ligands with high affinity and high specificity toward various targets has surged in the biomedical field in recent years. The traditional peptide screening procedure involves selection, sequencing, and characterization steps, and each step is manual and tedious. Herein, we developed a bimodal imprint microarray system to embrace the whole peptide screening process. Silver-sputtered silicon chip fabricated with microwell array can trap and pattern the candidate peptide beads in a one-well-one-bead manner. Peptides on beads were photocleaved in situ. A portion of the peptide in each well was transferred to a gold-coated chip to print the peptide array for high-throughput affinity analyses by surface plasmon resonance imaging (SPRi), and the peptide left in the silver-sputtered chip was ready for in situ single bead sequencing by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the bimodal imprint chip system, affinity peptides toward AHA were efficiently screened out from the 7 × 10(4) peptide library. The method provides a solution for high efficiency peptide screening.

Development of a Trypanosoma cruzi strain typing assay using MS2 peptide spectral libraries (Tc-STAMS2)

PubMed Central

de Oliveira, Gilberto Santos; Kawahara, Rebeca; Rosa-Fernandes, Livia; Avila, Carla Cristi; Teixeira, Marta M. G.; Larsen, Martin R.

2018-01-01

Background Chagas disease also known as American trypanosomiasis is caused by the protozoan Trypanosoma cruzi. Over the last 30 years, Chagas disease has expanded from a neglected parasitic infection of the rural population to an urbanized chronic disease, becoming a potentially emergent global health problem. T. cruzi strains were assigned to seven genetic groups (TcI-TcVI and TcBat), named discrete typing units (DTUs), which represent a set of isolates that differ in virulence, pathogenicity and immunological features. Indeed, diverse clinical manifestations (from asymptomatic to highly severe disease) have been attempted to be related to T.cruzi genetic variability. Due to that, several DTU typing methods have been introduced. Each method has its own advantages and drawbacks such as high complexity and analysis time and all of them are based on genetic signatures. Recently, a novel method discriminated bacterial strains using a peptide identification-free, genome sequence-independent shotgun proteomics workflow. Here, we aimed to develop a Trypanosoma cruzi Strain Typing Assay using MS/MS peptide spectral libraries, named Tc-STAMS2. Methods/Principal findings The Tc-STAMS2 method uses shotgun proteomics combined with spectral library search to assign and discriminate T. cruzi strains independently on the genome knowledge. The method is based on the construction of a library of MS/MS peptide spectra built using genotyped T. cruzi reference strains. For identification, the MS/MS peptide spectra of unknown T. cruzi cells are identified using the spectral matching algorithm SpectraST. The Tc-STAMS2 method allowed correct identification of all DTUs with high confidence. The method was robust towards different sample preparations, length of chromatographic gradients and fragmentation techniques. Moreover, a pilot inter-laboratory study showed the applicability to different MS platforms. Conclusions and significance This is the first study that develops a MS-based platform for T. cruzi strain typing. Indeed, the Tc-STAMS2 method allows T. cruzi strain typing using MS/MS spectra as discriminatory features and allows the differentiation of TcI-TcVI DTUs. Similar to genomic-based strategies, the Tc-STAMS2 method allows identification of strains within DTUs. Its robustness towards different experimental and biological variables makes it a valuable complementary strategy to the current T. cruzi genotyping assays. Moreover, this method can be used to identify DTU-specific features correlated with the strain phenotype. PMID:29608573

Development of a Trypanosoma cruzi strain typing assay using MS2 peptide spectral libraries (Tc-STAMS2).

PubMed

de Oliveira, Gilberto Santos; Kawahara, Rebeca; Rosa-Fernandes, Livia; Mule, Simon Ngao; Avila, Carla Cristi; Teixeira, Marta M G; Larsen, Martin R; Palmisano, Giuseppe

2018-04-01

Chagas disease also known as American trypanosomiasis is caused by the protozoan Trypanosoma cruzi. Over the last 30 years, Chagas disease has expanded from a neglected parasitic infection of the rural population to an urbanized chronic disease, becoming a potentially emergent global health problem. T. cruzi strains were assigned to seven genetic groups (TcI-TcVI and TcBat), named discrete typing units (DTUs), which represent a set of isolates that differ in virulence, pathogenicity and immunological features. Indeed, diverse clinical manifestations (from asymptomatic to highly severe disease) have been attempted to be related to T.cruzi genetic variability. Due to that, several DTU typing methods have been introduced. Each method has its own advantages and drawbacks such as high complexity and analysis time and all of them are based on genetic signatures. Recently, a novel method discriminated bacterial strains using a peptide identification-free, genome sequence-independent shotgun proteomics workflow. Here, we aimed to develop a Trypanosoma cruzi Strain Typing Assay using MS/MS peptide spectral libraries, named Tc-STAMS2. The Tc-STAMS2 method uses shotgun proteomics combined with spectral library search to assign and discriminate T. cruzi strains independently on the genome knowledge. The method is based on the construction of a library of MS/MS peptide spectra built using genotyped T. cruzi reference strains. For identification, the MS/MS peptide spectra of unknown T. cruzi cells are identified using the spectral matching algorithm SpectraST. The Tc-STAMS2 method allowed correct identification of all DTUs with high confidence. The method was robust towards different sample preparations, length of chromatographic gradients and fragmentation techniques. Moreover, a pilot inter-laboratory study showed the applicability to different MS platforms. This is the first study that develops a MS-based platform for T. cruzi strain typing. Indeed, the Tc-STAMS2 method allows T. cruzi strain typing using MS/MS spectra as discriminatory features and allows the differentiation of TcI-TcVI DTUs. Similar to genomic-based strategies, the Tc-STAMS2 method allows identification of strains within DTUs. Its robustness towards different experimental and biological variables makes it a valuable complementary strategy to the current T. cruzi genotyping assays. Moreover, this method can be used to identify DTU-specific features correlated with the strain phenotype.

Hunting for unexpected post-translational modifications by spectral library searching with tier-wise scoring.

PubMed

Ma, Chun Wai Manson; Lam, Henry

2014-05-02

Discovering novel post-translational modifications (PTMs) to proteins and detecting specific modification sites on proteins is one of the last frontiers of proteomics. At present, hunting for post-translational modifications remains challenging in widely practiced shotgun proteomics workflows due to the typically low abundance of modified peptides and the greatly inflated search space as more potential mass shifts are considered by the search engines. Moreover, most popular search methods require that the user specifies the modification(s) for which to search; therefore, unexpected and novel PTMs will not be detected. Here a new algorithm is proposed to apply spectral library searching to the problem of open modification searches, namely, hunting for PTMs without prior knowledge of what PTMs are in the sample. The proposed tier-wise scoring method intelligently looks for unexpected PTMs by allowing mass-shifted peak matches but only when the number of matches found is deemed statistically significant. This allows the search engine to search for unexpected modifications while maintaining its ability to identify unmodified peptides effectively at the same time. The utility of the method is demonstrated using three different data sets, in which the numbers of spectrum identifications to both unmodified and modified peptides were substantially increased relative to a regular spectral library search as well as to another open modification spectral search method, pMatch.

On the limited recognition of inorganic surfaces by short peptides compared with antibodies.

PubMed

Artzy-Schnirman, Arbel; Abu-Shah, Enas; Dishon, Matan; Soifer, Hadas; Sivan, Yotam; Reiter, Yoram; Benhar, Itai; Sivan, Uri

2014-06-01

The vast potential applications of biomolecules that bind inorganic surfaces led mostly to the isolation of short peptides that target selectively specific materials. The demonstrated differential affinity toward certain surfaces created the impression that the recognition capacity of short peptides may match that of rigid biomolecules. In the following, we challenge this view by comparing the capacity of antibody molecules to discriminate between the (100) and (111A) facets of a gallium arsenide semiconductor crystal with the capacity of short peptides to do the same. Applying selection from several peptide and single chain phage display libraries, we find a number of antibody molecules that bind preferentially a given crystal facet but fail to isolate, in dozens of attempts, a single peptide capable of such recognition. The experiments underscore the importance of rigidity to the recognition of inorganic flat targets and therefore set limitations on potential applications of short peptides in biomimetics. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Antiviral Peptides Targeting the West Nile Virus Envelope Protein▿

PubMed Central

Bai, Fengwei; Town, Terrence; Pradhan, Deepti; Cox, Jonathan; Ashish; Ledizet, Michel; Anderson, John F.; Flavell, Richard A.; Krueger, Joanna K.; Koski, Raymond A.; Fikrig, Erol

2007-01-01

West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 μM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 μM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses. PMID:17151121

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

PubMed

Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

2018-04-01

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library

PubMed Central

Adler, Adam S.; Bedinger, Daniel; Adams, Matthew S.; Asensio, Michael A.; Edgar, Robert C.; Leong, Renee; Leong, Jackson; Mizrahi, Rena A.; Spindler, Matthew J.; Bandi, Srinivasa Rao; Huang, Haichun; Brams, Peter; Johnson, David S.

2018-01-01

ABSTRACT Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs. PMID:29376776

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.

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage-display

PubMed Central

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K.

2012-01-01

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious and time consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13, the N-terminal Forkhead-associated domain (FHA1) of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be non-functional due to misfolding in the bacterial periplasm. To overcome this limitation, a library of FHA1 variants was constructed by mutagenic PCR and functional variants were isolated after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1-strand was discovered to be essential for phage-display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermal stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20–25 mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage-display. PMID:22985966

Systematic Testing of Belief-Propagation Estimates for Absolute Free Energies in Atomistic Peptides and Proteins.

PubMed

Donovan-Maiye, Rory M; Langmead, Christopher J; Zuckerman, Daniel M

2018-01-09

Motivated by the extremely high computing costs associated with estimates of free energies for biological systems using molecular simulations, we further the exploration of existing "belief propagation" (BP) algorithms for fixed-backbone peptide and protein systems. The precalculation of pairwise interactions among discretized libraries of side-chain conformations, along with representation of protein side chains as nodes in a graphical model, enables direct application of the BP approach, which requires only ∼1 s of single-processor run time after the precalculation stage. We use a "loopy BP" algorithm, which can be seen as an approximate generalization of the transfer-matrix approach to highly connected (i.e., loopy) graphs, and it has previously been applied to protein calculations. We examine the application of loopy BP to several peptides as well as the binding site of the T4 lysozyme L99A mutant. The present study reports on (i) the comparison of the approximate BP results with estimates from unbiased estimators based on the Amber99SB force field; (ii) investigation of the effects of varying library size on BP predictions; and (iii) a theoretical discussion of the discretization effects that can arise in BP calculations. The data suggest that, despite their approximate nature, BP free-energy estimates are highly accurate-indeed, they never fall outside confidence intervals from unbiased estimators for the systems where independent results could be obtained. Furthermore, we find that libraries of sufficiently fine discretization (which diminish library-size sensitivity) can be obtained with standard computing resources in most cases. Altogether, the extremely low computing times and accurate results suggest the BP approach warrants further study.

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage display.

PubMed

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K

2012-11-23

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious, and time-consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13 the N-terminal Forkhead-associated (FHA1) domain of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be nonfunctional due to misfolding in the bacterial periplasm. To overcome this limitation, we constructed a library of FHA1 variants by mutagenic PCR and isolated functional variants after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1 strand was discovered to be essential for phage display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermally stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20-25mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage display. Copyright © 2012 Elsevier Ltd. All rights reserved.

Experimental and Theoretical Approaches To Investigate the Immunogenicity of Taenia solium-Derived KE7 Antigen

PubMed Central

Bobes, Raúl J.; Navarrete-Perea, José; Ochoa-Leyva, Adrián; Anaya, Víctor Hugo; Hernández, Marisela; Cervantes-Torres, Jacquelynne; Estrada, Karel; Sánchez-Lopez, Filiberto; Soberón, Xavier; Rosas, Gabriela; Nunes, Cáris Maroni; García-Varela, Martín; Sotelo-Mundo, Rogerio Rafael; López-Zavala, Alonso Alexis; Gevorkian, Goar; Acero, Gonzalo; Laclette, Juan P.; Fragoso, Gladis

2017-01-01

ABSTRACT Taenia solium cysticercosis, a parasitic disease that affects human health in various regions of the world, is preventable by vaccination. Both the 97-amino-acid-long KETc7 peptide and its carboxyl-terminal, 18-amino-acid-long sequence (GK-1) are found in Taenia crassiceps. Both peptides have proven protective capacity against cysticercosis and are part of the highly conserved, cestode-native, 264-amino-acid long protein KE7. KE7 belongs to a ubiquitously distributed family of proteins associated with membrane processes and may participate in several vital cell pathways. The aim of this study was to identify the T. solium KE7 (TsKE7) full-length protein and to determine its immunogenic properties. Recombinant TsKE7 (rTsKE7) was expressed in Escherichia coli Rosetta2 cells and used to obtain mouse polyclonal antibodies. Anti-rTsKE7 antibodies detected the expected native protein among the 350 spots developed from T. solium cyst vesicular fluid in a mass spectrometry-coupled immune proteomic analysis. These antibodies were then used to screen a phage-displayed 7-random-peptide library to map B-cell epitopes. The recognized phages displayed 9 peptides, with the consensus motif Y(F/Y)PS sequence, which includes YYYPS (named GK-1M, for being a GK-1 mimotope), exactly matching a part of GK-1. GK-1M was recognized by 58% of serum samples from cysticercotic pigs with 100% specificity but induced weak protection against murine cysticercosis. In silico analysis revealed a universal T-cell epitope(s) in native TsKE7 potentially capable of stimulating cytotoxic T lymphocytes and helper T lymphocytes under different major histocompatibility complex class I and class II mouse haplotypes. Altogether, these results provide a rationale for the efficacy of the KETc7, rTsKE7, and GK-1 peptides as vaccines. PMID:28923896

Experimental and Theoretical Approaches To Investigate the Immunogenicity of Taenia solium-Derived KE7 Antigen.

PubMed

Bobes, Raúl J; Navarrete-Perea, José; Ochoa-Leyva, Adrián; Anaya, Víctor Hugo; Hernández, Marisela; Cervantes-Torres, Jacquelynne; Estrada, Karel; Sánchez-Lopez, Filiberto; Soberón, Xavier; Rosas, Gabriela; Nunes, Cáris Maroni; García-Varela, Martín; Sotelo-Mundo, Rogerio Rafael; López-Zavala, Alonso Alexis; Gevorkian, Goar; Acero, Gonzalo; Laclette, Juan P; Fragoso, Gladis; Sciutto, Edda

2017-12-01

Taenia solium cysticercosis, a parasitic disease that affects human health in various regions of the world, is preventable by vaccination. Both the 97-amino-acid-long KETc7 peptide and its carboxyl-terminal, 18-amino-acid-long sequence (GK-1) are found in Taenia crassiceps Both peptides have proven protective capacity against cysticercosis and are part of the highly conserved, cestode-native, 264-amino-acid long protein KE7. KE7 belongs to a ubiquitously distributed family of proteins associated with membrane processes and may participate in several vital cell pathways. The aim of this study was to identify the T. solium KE7 (TsKE7) full-length protein and to determine its immunogenic properties. Recombinant TsKE7 (rTsKE7) was expressed in Escherichia coli Rosetta2 cells and used to obtain mouse polyclonal antibodies. Anti-rTsKE7 antibodies detected the expected native protein among the 350 spots developed from T. solium cyst vesicular fluid in a mass spectrometry-coupled immune proteomic analysis. These antibodies were then used to screen a phage-displayed 7-random-peptide library to map B-cell epitopes. The recognized phages displayed 9 peptides, with the consensus motif Y(F/Y)PS sequence, which includes YYYPS (named GK-1M, for being a GK-1 mimotope), exactly matching a part of GK-1. GK-1M was recognized by 58% of serum samples from cysticercotic pigs with 100% specificity but induced weak protection against murine cysticercosis. In silico analysis revealed a universal T-cell epitope(s) in native TsKE7 potentially capable of stimulating cytotoxic T lymphocytes and helper T lymphocytes under different major histocompatibility complex class I and class II mouse haplotypes. Altogether, these results provide a rationale for the efficacy of the KETc7, rTsKE7, and GK-1 peptides as vaccines. Copyright © 2017 American Society for Microbiology.

A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches.

PubMed

Li, Chenxi; Liu, Hongyu; Li, Jinzhe; Liu, Dafei; Meng, Runze; Zhang, Qingshan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Tingting; Liu, Ming; Zhang, Yun

2016-01-01

Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.

Random and independent sampling of endogenous tryptic peptides from normal human EDTA plasma by liquid chromatography micro electrospray ionization and tandem mass spectrometry.

PubMed

Dufresne, Jaimie; Florentinus-Mefailoski, Angelique; Ajambo, Juliet; Ferwa, Ammara; Bowden, Peter; Marshall, John

2017-01-01

Normal human EDTA plasma samples were collected on ice, processed ice cold, and stored in a freezer at - 80 °C prior to experiments. Plasma test samples from the - 80 °C freezer were thawed on ice or intentionally warmed to room temperature. Protein content was measured by CBBR binding and the release of alcohol soluble amines by the Cd ninhydrin assay. Plasma peptides released over time were collected over C18 for random and independent sampling by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) and correlated with X!TANDEM. Fully tryptic peptides by X!TANDEM returned a similar set of proteins, but was more computationally efficient, than "no enzyme" correlations. Plasma samples maintained on ice, or ice with a cocktail of protease inhibitors, showed lower background amounts of plasma peptides compared to samples incubated at room temperature. Regression analysis indicated that warming plasma to room temperature, versus ice cold, resulted in a ~ twofold increase in the frequency of peptide identification over hours-days of incubation at room temperature. The type I error rate of the protein identification from the X!TANDEM algorithm combined was estimated to be low compared to a null model of computer generated random MS/MS spectra. The peptides of human plasma were identified and quantified with low error rates by random and independent sampling that revealed 1000s of peptides from hundreds of human plasma proteins from endogenous tryptic peptides.

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, J.; Varner, J.E.

1985-07-01

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 asmore » a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.« less

Genetic Selection of Peptide Aptamers That Interact and Inhibit Both Small Protein B and Alternative Ribosome-Rescue Factor A of Aeromonas veronii C4.

PubMed

Liu, Peng; Chen, Yong; Wang, Dan; Tang, Yanqiong; Tang, Hongqian; Song, Haichao; Sun, Qun; Zhang, Yueling; Liu, Zhu

2016-01-01

Aeromonas veronii is a pathogenic gram-negative bacterium, which infects a variety of animals and results in mass mortality. The stalled-ribosome rescues are reported to ensure viability and virulence under stress conditions, of which primarily include trans-translation and alternative ribosome-rescue factor A (ArfA) in A. veronii. For identification of specific peptides that interact and inhibit the stalled-ribosome rescues, peptide aptamer library (pTRG-SN-peptides) was constructed using pTRG as vector and Staphylococcus aureus nuclease (SN) as scaffold protein, in which 16 random amino acids were introduced to form an exposed surface loop. In the meantime both Small Protein B (SmpB) which acts as one of the key components in trans-translation, and ArfA were inserted to pBT to constitute pBT-SmpB and pBT-ArfA, respectively. The peptide aptamer PA-2 was selected from pTRG-SN-peptides by bacterial two-hybrid system (B2H) employing pBT-SmpB or pBT-ArfA as baits. The conserved sites G133K134 and D138K139R140 of C-terminal SmpB were identified by interacting with N-terminal SN, and concurrently the residue K62 of ArfA was recognized by interacting with the surface loop of the specific peptide aptamer PA-2. The expression plasmids pN-SN or pN-PA-2, which combined the duplication origin of pRE112 with the neokanamycin promoter expressing SN or PA-2, were created and transformed into A. veronii C4, separately. The engineered A. veronii C4 which endowing SN or PA-2 expression impaired growth capabilities under stress conditions including temperatures, sucrose, glucose, potassium chloride (KCl) and antibiotics, and the stress-related genes rpoS and nhaP were down-regulated significantly by Quantitative Real-time PCR (qRT-PCR) when treating in 2.0% KCl. Thus, the engineered A. veronii C4 conferring PA-2 expression might be potentially attenuated vaccine, and also the peptide aptamer PA-2 could develop as anti-microbial drugs targeted to the ribosome rescued factors in A. veronii.

Genetic Selection of Peptide Aptamers That Interact and Inhibit Both Small Protein B and Alternative Ribosome-Rescue Factor A of Aeromonas veronii C4

PubMed Central

Liu, Peng; Chen, Yong; Wang, Dan; Tang, Yanqiong; Tang, Hongqian; Song, Haichao; Sun, Qun; Zhang, Yueling; Liu, Zhu

2016-01-01

Aeromonas veronii is a pathogenic gram-negative bacterium, which infects a variety of animals and results in mass mortality. The stalled-ribosome rescues are reported to ensure viability and virulence under stress conditions, of which primarily include trans-translation and alternative ribosome-rescue factor A (ArfA) in A. veronii. For identification of specific peptides that interact and inhibit the stalled-ribosome rescues, peptide aptamer library (pTRG-SN-peptides) was constructed using pTRG as vector and Staphylococcus aureus nuclease (SN) as scaffold protein, in which 16 random amino acids were introduced to form an exposed surface loop. In the meantime both Small Protein B (SmpB) which acts as one of the key components in trans-translation, and ArfA were inserted to pBT to constitute pBT-SmpB and pBT-ArfA, respectively. The peptide aptamer PA-2 was selected from pTRG-SN-peptides by bacterial two-hybrid system (B2H) employing pBT-SmpB or pBT-ArfA as baits. The conserved sites G133K134 and D138K139R140 of C-terminal SmpB were identified by interacting with N-terminal SN, and concurrently the residue K62 of ArfA was recognized by interacting with the surface loop of the specific peptide aptamer PA-2. The expression plasmids pN-SN or pN-PA-2, which combined the duplication origin of pRE112 with the neokanamycin promoter expressing SN or PA-2, were created and transformed into A. veronii C4, separately. The engineered A. veronii C4 which endowing SN or PA-2 expression impaired growth capabilities under stress conditions including temperatures, sucrose, glucose, potassium chloride (KCl) and antibiotics, and the stress-related genes rpoS and nhaP were down-regulated significantly by Quantitative Real-time PCR (qRT-PCR) when treating in 2.0% KCl. Thus, the engineered A. veronii C4 conferring PA-2 expression might be potentially attenuated vaccine, and also the peptide aptamer PA-2 could develop as anti-microbial drugs targeted to the ribosome rescued factors in A. veronii. PMID:27588015

Human recombinant Fab fragment from combinatorial libraries of a B-cell lymphoma patient recognizes core protein of chondroitin sulphate proteoglycan 4.

PubMed

Egami, Yoko; Narushima, Yuta; Ohshima, Motohiro; Yoshida, Akira; Yoneta, Naruki; Masaki, Yasufumi; Itoh, Kunihiko

2018-01-01

CD antigens are well known as therapeutic targets of B-cell lymphoma. To isolate therapeutic antibodies that recognize novel targets other than CD antigens, we constructed a phage display combinatorial antibody Fab library from bone marrow lymphocytes of B-cell lymphoma patient. To eliminate antibodies reactive with known B-cell lymphoma antigen, non-hematopoietic and patient's sera reactive HeLaS3 cells was selected as a target of whole cell panning. Five rounds of panning against live HeLaS3 cells retrieved single Fab clone, termed AHSA (Antibody to HeLa Surface Antigen). Using phage display random peptide library, LSYLEP was identified as an epitope sequence of AHSA. LC-MS/MS analysis of AHSA-precipitated HeLaS3 cell lysates detected several fragments corresponding to the sequence of chondroitin sulphate proteoglycan 4 (CSPG4) core protein. Since LSYLEP sequence was at the position of 313-318 of CSPG4, we considered that CSPG4 was AHSA-associated antigen. Double staining of CSPG4-postive MDA-MB-435S cells with AHSA and anti-CSPG4 rabbit antibody showed identical staining position, and reduced AHSA reactivity was observed in CSPG4-siRNA treated MDA-MB-435S cells. In conclusion, we retrieved a human Fab from antibody library of B-cell lymphoma patient, and identified CSPG4 as a recognizing antigen. AHSA may have potential benefits for development of CSPG4-targeting theranostics for B-cell lymphoma. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Identification and characterization of antimicrobial peptide, defensin, in the taiga tick, Ixodes persulcatus.

PubMed

Saito, Y; Konnai, S; Yamada, S; Imamura, S; Nishikado, H; Ito, T; Onuma, M; Ohashi, K

2009-08-01

Ixodes persulcatus is the primary vector for human tick-borne diseases in Japan. A cDNA library was constructed from whole body homogenates of fed nymphs of I. persulcatus. From this library, one cDNA encoding defensin-like antimicrobial peptide was identified. The amino-acid sequence showed high similarity to those of the defensins of other ticks and arthropods. I. persulcatus defensin mRNA transcripts were detected at all life cycle stages of fed ticks and found to be predominantly expressed in the midguts of adult female ticks, but not in the salivary glands, a finding corroborated by Western blotting analysis. To investigate the function of I. persulcatus defensin, we examined its antibacterial activity by evaluation of growth of several bacterial strains in the presence of the synthetic peptide. The defensin from I. persulcatus markedly inhibited the growth of Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis and Corynebacterium renale, but not Gram-negative bacteria except Escherichia coli O157. In conclusion, these results suggest that I. persulcatus defensin may be playing a significant role in the defence against microbes from bloodmeals.

MaRiMba: a software application for spectral library-based MRM transition list assembly.

PubMed

Sherwood, Carly A; Eastham, Ashley; Lee, Lik Wee; Peterson, Amelia; Eng, Jimmy K; Shteynberg, David; Mendoza, Luis; Deutsch, Eric W; Risler, Jenni; Tasman, Natalie; Aebersold, Ruedi; Lam, Henry; Martin, Daniel B

2009-10-01

Multiple reaction monitoring mass spectrometry (MRM-MS) is a targeted analysis method that has been increasingly viewed as an avenue to explore proteomes with unprecedented sensitivity and throughput. We have developed a software tool, called MaRiMba, to automate the creation of explicitly defined MRM transition lists required to program triple quadrupole mass spectrometers in such analyses. MaRiMba creates MRM transition lists from downloaded or custom-built spectral libraries, restricts output to specified proteins or peptides, and filters based on precursor peptide and product ion properties. MaRiMba can also create MRM lists containing corresponding transitions for isotopically heavy peptides, for which the precursor and product ions are adjusted according to user specifications. This open-source application is operated through a graphical user interface incorporated into the Trans-Proteomic Pipeline, and it outputs the final MRM list to a text file for upload to MS instruments. To illustrate the use of MaRiMba, we used the tool to design and execute an MRM-MS experiment in which we targeted the proteins of a well-defined and previously published standard mixture.

First venom gland transcriptomic analysis of Iranian yellow scorpion "Odonthubuthus doriae" with some new findings.

PubMed

NaderiSoorki, Maryam; Galehdari, Hamid; Baradaran, Masomeh; Jalali, Amir

2016-09-15

Scorpion venom contains mixture of biologic molecules including selective toxins with medical capability. Odonthubuthus doriae (O. doriae) belonged to Buthidae family of scorpions and gained more interest among Iranian dangerous scorpion since 2005. We constructed the first cDNA library to explore the transcriptomic composition of this Iranian scorpiontelson. Then by used of bioinformatic software each expression sequence taq (EST) from the library analyzed and its quiddity was clear. Analysis showed that toxins (42%) had more venom transcript than other component such as antimicrobial peptides, venom peptides and cell proteins. Over 16% of transcripts didn't have any open reading frames (ORF), however their sequences showed similarity by other scorpion sequences. One EST didn't have any similarity by known scorpion peptides. For the first time; we report a comprehensive study of an Iranian scorpion with interesting and novel findings. We characterized a new putative sodium channel modifier in scorpions by some bioinformatics software, and then predicted its structure and function. Copyright © 2016. Published by Elsevier Ltd.

Simultaneous alignment and clustering of peptide data using a Gibbs sampling approach.

PubMed

Andreatta, Massimo; Lund, Ole; Nielsen, Morten

2013-01-01

Proteins recognizing short peptide fragments play a central role in cellular signaling. As a result of high-throughput technologies, peptide-binding protein specificities can be studied using large peptide libraries at dramatically lower cost and time. Interpretation of such large peptide datasets, however, is a complex task, especially when the data contain multiple receptor binding motifs, and/or the motifs are found at different locations within distinct peptides. The algorithm presented in this article, based on Gibbs sampling, identifies multiple specificities in peptide data by performing two essential tasks simultaneously: alignment and clustering of peptide data. We apply the method to de-convolute binding motifs in a panel of peptide datasets with different degrees of complexity spanning from the simplest case of pre-aligned fixed-length peptides to cases of unaligned peptide datasets of variable length. Example applications described in this article include mixtures of binders to different MHC class I and class II alleles, distinct classes of ligands for SH3 domains and sub-specificities of the HLA-A*02:01 molecule. The Gibbs clustering method is available online as a web server at http://www.cbs.dtu.dk/services/GibbsCluster.

Cholesterol-directed nanoparticle assemblies based on single amino acid peptide mutations activate cellular uptake and decrease tumor volume† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc02616a Click here for additional data file.

PubMed Central

Li, Shang; Zou, Rongfeng; Tu, Yaoquan

2017-01-01

Peptide drugs have been difficult to translate into effective therapies due to their low in vivo stability. Here, we report a strategy to develop peptide-based therapeutic nanoparticles by screening a peptide library differing by single-site amino acid mutations of lysine-modified cholesterol. Certain cholesterol-modified peptides are found to promote and stabilize peptide α-helix formation, resulting in selectively cell-permeable peptides. One cholesterol-modified peptide self-assembles into stable nanoparticles with considerable α-helix propensity stabilized by intermolecular van der Waals interactions between inter-peptide cholesterol molecules, and shows 68.3% stability after incubation with serum for 16 h. The nanoparticles in turn interact with cell membrane cholesterols that are disproportionately present in cancer cell membranes, inducing lipid raft-mediated endocytosis and cancer cell death. Our results introduce a strategy to identify peptide nanoparticles that can effectively reduce tumor volumes when administered to in in vivo mice models. Our results also provide a simple platform for developing peptide-based anticancer drugs. PMID:29163910

Identification and characterization of epitopes on Plasmodium knowlesi merozoite surface protein-142 (MSP-142) using synthetic peptide library and phage display library.

PubMed

Cheong, Fei Wen; Fong, Mun Yik; Lau, Yee Ling

2016-02-01

Plasmodium knowlesi can cause potentially life threatening human malaria. The Plasmodium merozoite surface protein-142 (MSP-142) is a potential target for malaria blood stage vaccine, and for diagnosis of malaria. Two epitope mapping techniques were used to identify the potential epitopes within P. knowlesi MSP-142. Nine and 14 potential epitopes were identified using overlapping synthetic peptide library and phage display library, respectively. Two regions on P. knowlesi MSP-142 (amino acid residues 37-95 and residues 240-289) were identified to be the potential dominant epitope regions. Two of the prominent epitopes, P10 (TAKDGMEYYNKMGELYKQ) and P31 (RCLLGFKEVGGKCVPASI), were evaluated using mouse model. P10- and P31-immunized mouse sera reacted with recombinant P. knowlesi MSP-142, with the IgG isotype distribution of IgG2b>IgG1>IgG2a>IgG3. Significant higher level of cytokines interferon-gamma and interleukin-2 was detected in P31-immunized mice. Both P10 and P31 could be the suitable epitope candidates to be used in malaria vaccine designs and immunodiagnostic assays, provided further evaluation is needed to validate the potential uses of these epitopes. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica.

PubMed

Zhong, Jian; Wang, Wenhong; Yang, Xiaomei; Yan, Xiuwen; Liu, Rui

2013-01-01

Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

A general strategy for synthesis of cyclophane-braced peptide macrocycles via palladium-catalysed intramolecular sp3 C-H arylation

NASA Astrophysics Data System (ADS)

Zhang, Xuekai; Lu, Gang; Sun, Meng; Mahankali, Madhu; Ma, Yanfei; Zhang, Mingming; Hua, Wangde; Hu, Yuting; Wang, Qingbing; Chen, Jinghuo; He, Gang; Qi, Xiangbing; Shen, Weijun; Liu, Peng; Chen, Gong

2018-05-01

New methods capable of effecting cyclization, and forming novel three-dimensional structures while maintaining favourable physicochemical properties are needed to facilitate the development of cyclic peptide-based drugs that can engage challenging biological targets, such as protein-protein interactions. Here, we report a highly efficient and generally applicable strategy for constructing new types of peptide macrocycles using palladium-catalysed intramolecular C(sp3)-H arylation reactions. Easily accessible linear peptide precursors of simple and versatile design can be selectively cyclized at the side chains of either aromatic or modified non-aromatic amino acid units to form various cyclophane-braced peptide cycles. This strategy provides a powerful tool to address the long-standing challenge of size- and composition-dependence in peptide macrocyclization, and generates novel peptide macrocycles with uniquely buttressed backbones and distinct loop-type three-dimensional structures. Preliminary cell proliferation screening of the pilot library revealed a potent lead compound with selective cytotoxicity toward proliferative Myc-dependent cancer cell lines.

A phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments.

PubMed

Solforosi, Laura; Mancini, Nicasio; Canducci, Filippo; Clementi, Nicola; Sautto, Giuseppe Andrea; Diotti, Roberta Antonia; Clementi, Massimo; Burioni, Roberto

2012-07-01

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III and light chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide, whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was used to generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragment antibody directed against the nucleoprotein (NP) of Influenza A virus.

Host Defense Antimicrobial Peptides as Antibiotics: Design and Application Strategies

PubMed Central

Mishra, Biswajit; Reiling, Scott; Zarena, D.; Wang, Guangshun

2017-01-01

This review deals with the design and application strategies of new antibiotics based on naturally occurring antimicrobial peptides (AMPs). The initial candidate can be designed based on three-dimensional structure or selected from a library of peptides from natural or laboratory sources followed by optimization via structure-activity relationship studies. There are also advanced application strategies such as induction of AMP expression from host cells by various factors (e.g., metals, amino acids, vitamin D and sunlight), the use of engineered probiotic bacteria to deliver peptides, the design of prodrug and peptide conjugates to improve specific targeting. In addition, combined uses of newly developed AMPs with existing antimicrobial agents may provide a practical avenue for effective management of antibiotic-resistant bacteria (superbugs, including biofilm). Finally, we highlight AMPs already in use or under clinical trials. PMID:28399505

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.

Economical analysis of saturation mutagenesis experiments

PubMed Central

Acevedo-Rocha, Carlos G.; Reetz, Manfred T.; Nov, Yuval

2015-01-01

Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced. PMID:26190439

Collision-Induced Dissociation of Deprotonated Peptides. Relative Abundance of Side-Chain Neutral Losses, Residue-Specific Product Ions, and Comparison with Protonated Peptides

NASA Astrophysics Data System (ADS)

Liang, Yuxue; Neta, Pedatsur; Yang, Xiaoyu; Stein, Stephen E.

2018-03-01

High-accuracy MS/MS spectra of deprotonated ions of 390 dipeptides and 137 peptides with three to six residues are studied. Many amino acid residues undergo neutral losses from their side chains. The most abundant is the loss of acetaldehyde from threonine. The abundance of losses from the side chains of other amino acids is estimated relative to that of threonine. While some amino acids lose the whole side chain, others lose only part of it, and some exhibit two or more different losses. Side-chain neutral losses are less abundant in the spectra of protonated peptides, being significant mainly for methionine and arginine. In addition to the neutral losses, many amino acid residues in deprotonated peptides produce specific negative ions after peptide bond cleavage. An expanded list of fragment ions from protonated peptides is also presented and compared with those of deprotonated peptides. Fragment ions are mostly different for these two cases. These lists of fragments are used to annotate peptide mass spectral libraries and to aid in the confirmation of specific amino acids in peptides. [Figure not available: see fulltext.

Collision-Induced Dissociation of Deprotonated Peptides. Relative Abundance of Side-Chain Neutral Losses, Residue-Specific Product Ions, and Comparison with Protonated Peptides.

PubMed

Liang, Yuxue; Neta, Pedatsur; Yang, Xiaoyu; Stein, Stephen E

2018-03-01

High-accuracy MS/MS spectra of deprotonated ions of 390 dipeptides and 137 peptides with three to six residues are studied. Many amino acid residues undergo neutral losses from their side chains. The most abundant is the loss of acetaldehyde from threonine. The abundance of losses from the side chains of other amino acids is estimated relative to that of threonine. While some amino acids lose the whole side chain, others lose only part of it, and some exhibit two or more different losses. Side-chain neutral losses are less abundant in the spectra of protonated peptides, being significant mainly for methionine and arginine. In addition to the neutral losses, many amino acid residues in deprotonated peptides produce specific negative ions after peptide bond cleavage. An expanded list of fragment ions from protonated peptides is also presented and compared with those of deprotonated peptides. Fragment ions are mostly different for these two cases. These lists of fragments are used to annotate peptide mass spectral libraries and to aid in the confirmation of specific amino acids in peptides. Graphical Abstract ᅟ.

Peptides for functionalization of InP semiconductors.

PubMed

Estephan, Elias; Saab, Marie-belle; Larroque, Christian; Martin, Marta; Olsson, Fredrik; Lourdudoss, Sebastian; Gergely, Csilla

2009-09-15

The challenge is to achieve high specificity in molecular sensing by proper functionalization of micro/nano-structured semiconductors by peptides that reveal specific recognition for these structures. Here we report on surface modification of the InP semiconductors by adhesion peptides produced by the phage display technique. An M13 bacteriophage library has been used to screen 10(10) different peptides against the InP(001) and the InP(111) surfaces to finally isolate specific peptides for each orientation of the InP. MALDI-TOF/TOF mass spectrometry has been employed to study real affinity of the peptide towards the InP surfaces. The peptides serve for controlled placement of biotin onto InP to bind then streptavidin. Our Atomic Force Microscopy study revealed a total surface coverage of molecules when the InP surface was functionalized by its specific biotinylated peptide (YAIKGPSHFRPS). Finally, fluorescence microscopy has been employed to demonstrate the preferential attachment of the peptide onto a micro-patterned InP surface. Use of substrate specific peptides could present an alternative solution for the problems encountered in the actually existing sensing methods and molecular self-assembly due to the unwanted unspecific interactions.

Using peptide array to identify binding motifs and interaction networks for modular domains.

PubMed

Li, Shawn S-C; Wu, Chenggang

2009-01-01

Specific protein-protein interactions underlie all essential biological processes and form the basis of cellular signal transduction. The recognition of a short, linear peptide sequence in one protein by a modular domain in another represents a common theme of macromolecular recognition in cells, and the importance of this mode of protein-protein interaction is highlighted by the large number of peptide-binding domains encoded by the human genome. This phenomenon also provides a unique opportunity to identify protein-protein binding events using peptide arrays and complementary biochemical assays. Accordingly, high-density peptide array has emerged as a useful tool by which to map domain-mediated protein-protein interaction networks at the proteome level. Using the Src-homology 2 (SH2) and 3 (SH3) domains as examples, we describe the application of oriented peptide array libraries in uncovering specific motifs recognized by an SH2 domain and the use of high-density peptide arrays in identifying interaction networks mediated by the SH3 domain. Methods reviewed here could also be applied to other modular domains, including catalytic domains, that recognize linear peptide sequences.

Theoretical design of a new chimeric protein for the treatment of breast cancer

PubMed Central

Soleimani, Meysam; Mahnam, Karim; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

2016-01-01

p28 and NRC peptides are two anticancer peptides with various mechanisms have shown to be effective against breast cancer. Therefore, it seems that construction of a chimeric protein containing the two peptides might cause synergistic cytotoxic effects. However, since the two peptides bear opposite charges, production of a chimeric protein in which the two moieties do not intervene each other is difficult. In this study, our goal was to find a suitable peptide linker for the new chimeric protein in a manner that none of the peptides intervene the other’s function. We selected some linkers with different characteristics and lengths and created a small library of the chimeric proteins harboring these linkers. Homology modeling and molecular dynamic simulation revealed that (PA)5P and (EAAAK)3 linkers can separate the p28 and NRC peptides effectively. Thus, the chimeric protein linked with (PA)5P or (EAAAK)3 linkers might show synergistic and stronger anticancer effects than the separate peptide moieties because they could exert their cytotoxic effects freely which is not influenced by the other part. PMID:27499788

Anticancer activities of bovine and human lactoferricin-derived peptides.

PubMed

Arias, Mauricio; Hilchie, Ashley L; Haney, Evan F; Bolscher, Jan G M; Hyndman, M Eric; Hancock, Robert E W; Vogel, Hans J

2017-02-01

Lactoferrin (LF) is a mammalian host defense glycoprotein with diverse biological activities. Peptides derived from the cationic region of LF possess cytotoxic activity against cancer cells in vitro and in vivo. Bovine lactoferricin (LFcinB), a peptide derived from bovine LF (bLF), exhibits broad-spectrum anticancer activity, while a similar peptide derived from human LF (hLF) is not as active. In this work, several peptides derived from the N-terminal regions of bLF and hLF were studied for their anticancer activities against leukemia and breast-cancer cells, as well as normal peripheral blood mononuclear cells. The cyclized LFcinB-CLICK peptide, which possesses a stable triazole linkage, showed improved anticancer activity, while short peptides hLF11 and bLF10 were not cytotoxic to cancer cells. Interestingly, hLF11 can act as a cell-penetrating peptide; when combined with the antimicrobial core sequence of LFcinB (RRWQWR) through either a Pro or Gly-Gly linker, toxicity to Jurkat cells increased. Together, our work extends the library of LF-derived peptides tested for anticancer activity, and identified new chimeric peptides with high cytotoxicity towards cancerous cells. Additionally, these results support the notion that short cell-penetrating peptides and antimicrobial peptides can be combined to create new adducts with increased potency.

Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

PubMed

Zhang, Limeng; Zhou, Xue; Fan, Ziyao; Tang, Wei; Chen, Liang; Dai, Jian; Wei, Yuhua; Zhang, Jianxin; Yang, Xuan; Yang, Xijing; Liu, Daolong; Yu, Liquan; Zhang, Hua; Wu, Zhijun; Yu, Yongzhong; Sun, Hunan; Cui, Yudong

2015-01-01

Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics

NASA Astrophysics Data System (ADS)

Woo, Sun Young; Lee, Hwankyu

2016-03-01

Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect.

All-atom simulations and free-energy calculations of coiled-coil peptides with lipid bilayers: binding strength, structural transition, and effect on lipid dynamics.

PubMed

Woo, Sun Young; Lee, Hwankyu

2016-03-01

Peptides E and K, which are synthetic coiled-coil peptides for membrane fusion, were simulated with lipid bilayers composed of lipids and cholesterols at different ratios using all-atom models. We first calculated free energies of binding from umbrella sampling simulations, showing that both E and K peptides tend to adsorb onto the bilayer surface, which occurs more strongly in the bilayer composed of smaller lipid headgroups. Then, unrestrained simulations show that K peptides more deeply insert into the bilayer with partially retaining the helical structure, while E peptides less insert and predominantly become random coils, indicating the structural transition from helices to random coils, in quantitative agreement with experiments. This is because K peptides electrostatically interact with lipid phosphates, as well as because hydrocarbons of lysines of K peptide are longer than those of glutamic acids of E peptide and thus form stronger hydrophobic interactions with lipid tails. This deeper insertion of K peptide increases the bilayer dynamics and a vacancy below the peptide, leading to the rearrangement of smaller lipids. These findings help explain the experimentally observed or proposed differences in the insertion depth, binding strength, and structural transition of E and K peptides, and support the snorkeling effect.

Effects of Teaching a Library Usage Unit to Seventh Graders.

ERIC Educational Resources Information Center

Gifford, Vernon; Gifford, Jean

This document reports the results of a study undertaken to determine if teaching a 2-week unit on library usage to 7th graders would increase their use of the library. From three non-ability grouped 7th grade classes, two classes of 26 students each were randomly selected for the study. One class was randomly assigned as the experimental group and…

Key Aspects of Nucleic Acid Library Design for in Vitro Selection

PubMed Central

Vorobyeva, Maria A.; Davydova, Anna S.; Vorobjev, Pavel E.; Pyshnyi, Dmitrii V.; Venyaminova, Alya G.

2018-01-01

Nucleic acid aptamers capable of selectively recognizing their target molecules have nowadays been established as powerful and tunable tools for biospecific applications, be it therapeutics, drug delivery systems or biosensors. It is now generally acknowledged that in vitro selection enables one to generate aptamers to almost any target of interest. However, the success of selection and the affinity of the resulting aptamers depend to a large extent on the nature and design of an initial random nucleic acid library. In this review, we summarize and discuss the most important features of the design of nucleic acid libraries for in vitro selection such as the nature of the library (DNA, RNA or modified nucleotides), the length of a randomized region and the presence of fixed sequences. We also compare and contrast different randomization strategies and consider computer methods of library design and some other aspects. PMID:29401748

An open-source computational and data resource to analyze digital maps of immunopeptidomes

DOE PAGES

Caron, Etienne; Espona, Lucia; Kowalewski, Daniel J.; ...

2015-07-08

We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.

Artificial neural network study on organ-targeting peptides

NASA Astrophysics Data System (ADS)

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.

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

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.

Production of a novel camel single-domain antibody specific for the type III mutant EGFR.

PubMed

Omidfar, K; Rasaee, M J; Modjtahedi, H; Forouzandeh, M; Taghikhani, M; Golmakani, N

2004-01-01

Camelids have a unique immune system capable of producing single-domain heavy-chain antibodies. The antigen-specific domain of these heavy-chain IgGs (VHH) are the smallest binding units produced by the immune system. In this study, we report the isolation and characterization of several binders against the epidermal growth factor receptor (EGFR) vIII retrieved from immune library of camels (Camelus bactrianus and Camelus dromedarius). The EGFRvIII is a ligand-independent, constitutively active, mutated form of the wild-type EGFR. The expression of EGFRvIII has been demonstrated in a wide range of human malignancies, including gliomas, and breast, prostate, ovarian and lung cancer. Camels were immunized with a synthetic peptide corresponding to a mutated sequence and tissue homogenates. Single-domain antibodies (VHH) were directly selected by panning a phage display library on successively decreasing amounts of synthetic peptide immobilized on magnetic beads. The anti-EGFRvIII camel single-domain antibodies selectively bound to the EGFRvIII peptide and reacted specifically with the immunoaffinity-purified antigen from a non-small cell lung cancer patient. These antibodies with affinities in the nanomolar range recognized the EGFRvIII peptide and affinity-purified mutated receptor. We concluded that using the phage display technique, antigen-specific VHH antibody fragments are readily accessible from the camelids. These antibodies may be good candidates for tumor-diagnostic and therapeutic applications. Copyright 2004 S. Karger AG, Basel.

The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase

PubMed Central

Bossa, Francesco; Barra, Donatella; Carloni, Massimo; Fasella, Paolo; Riva, Francesca; Doonan, Shawn; Doonan, Hilary J.; Hanford, Robin; Vernon, Charles A.; Walker, John M.

1973-01-01

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. PMID:4748834

In silico design of smart binders to anthrax PA

NASA Astrophysics Data System (ADS)

Sellers, Michael; Hurley, Margaret M.

2012-06-01

The development of smart peptide binders requires an understanding of the fundamental mechanisms of recognition which has remained an elusive grail of the research community for decades. Recent advances in automated discovery and synthetic library science provide a wealth of information to probe fundamental details of binding and facilitate the development of improved models for a priori prediction of affinity and specificity. Here we present the modeling portion of an iterative experimental/computational study to produce high affinity peptide binders to the Protective Antigen (PA) of Bacillus anthracis. The result is a general usage, HPC-oriented, python-based toolkit based upon powerful third-party freeware, which is designed to provide a better understanding of peptide-protein interactions and ultimately predict and measure new smart peptide binder candidates. We present an improved simulation protocol with flexible peptide docking to the Anthrax Protective Antigen, reported within the context of experimental data presented in a companion work.

PRO_LIGAND: An approach to de novo molecular design. 4. Application to the design of peptides

NASA Astrophysics Data System (ADS)

Frenkel, David; Clark, David E.; Li, Jin; Murray, Christopher W.; Robson, Barry; Waszkowycz, Bohdan; Westhead, David R.

1995-06-01

In some instances, peptides can play an important role in the discovery of lead compounds. This paper describes the peptide design facility of the de novo drug design package, PRO_LIGAND. The package provides a unified framework for the design of peptides that are similar or complementary to a specified target. The approach uses single amino acid residues, selected from preconstructed libraries of different residues and conformations, and places them on top of predefined target interaction sites. This approach is a well-tested methodology for the design of organics but has not been used for peptides before. Peptides represent a difficulty because of their great conformational flexibility and a study of the advantages and disavantages of this simple approach is an important step in the development of design tools. After a description of our general approach, a more detailed discussion of its adaptation to peptides is given. The method is then applied to the design of peptide-based inhibitors to HIV-1 protease and the design of structural mimics of the surface region of lysozyme. The results are encouraging and point the way towards further development of interaction site-based approaches for peptide design.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Liu, Yang; Zu, Xiangyang

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.more » 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.« less

Engineering peptide ligase specificity by proteomic identification of ligation sites.

PubMed

Weeks, Amy M; Wells, James A

2018-01-01

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

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

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

USDA-ARS?s Scientific Manuscript database

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...

Randomized Multicenter Trial of the Effects of Melanoma-Associated Helper Peptides and Cyclophosphamide on the Immunogenicity of a Multipeptide Melanoma Vaccine

PubMed Central

Slingluff, Craig L.; Petroni, Gina R.; Chianese-Bullock, Kimberly A.; Smolkin, Mark E.; Ross, Merrick I.; Haas, Naomi B.; von Mehren, Margaret; Grosh, William W.

2011-01-01

Purpose This multicenter randomized trial was designed to test whether melanoma-associated helper peptides augment CD8+ T-cell responses to a melanoma vaccine and whether cyclophosphamide (CY) pretreatment augments CD4+ or CD8+ T-cell responses to that vaccine. Patients and Methods In all, 167 eligible patients with resected stage IIB to IV melanoma were randomly assigned to four vaccination study arms. Patients were vaccinated with 12 class I major histocompatibility complex–restricted melanoma peptides (12MP) to stimulate CD8+ T cells and were randomly assigned to receive a tetanus helper peptide or a mixture of six melanoma-associated helper peptides (6MHP) to stimulate CD4+ T cells. Before vaccination, patients were also randomly assigned to receive CY pretreatment or not. T-cell responses were assessed by an ex vivo interferon gamma ELISpot assay. Clinical outcomes and toxicities were recorded. Results Vaccination with 12MP plus tetanus induced CD8+ T-cell responses in 78% of patients and CD4+ T-cell responses to tetanus peptide in 93% of patients. Vaccination with 12MP plus 6MHP induced CD8+ responses in 19% of patients and CD4+ responses to 6MHP in 48% of patients. CY had no significant effect on T-cell responses. Overall 3-year survival was 79% (95% CI, 71% to 86%), with no significant differences (at this point) by study arm. Conclusion Melanoma-associated helper peptides paradoxically decreased CD8+ T-cell responses to a melanoma vaccine (P < .001), and CY pretreatment had no immunologic or clinical effect. Prior work showed immunologic and clinical activity of 6MHP alone. Possible explanations for negative effects on CD8 responses include modulation of homing receptor expression or induction of antigen-specific regulatory T cells. PMID:21690475

Toxin Inhibition - Deconvolution Strategies and Assay Screening of Combinatorial Peptide Libraries

DTIC Science & Technology

2007-08-01

that could serve as lead compounds in the development of drug therapies to toxins. The libraries have typical structures of X I - X2 - hinge - X3 - X4...or passive vaccines have limited efficacies. There are no drug prophylaxes or therapies available. This technical report describes research to provide...broadly applicable drug discovery methods for a wide range of toxins. Future Work: The work conducted in the TIF project raised and renewed interest in

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.…

Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

NASA Astrophysics Data System (ADS)

Oda, Akifumi; Fukuyoshi, Shuichi

2015-06-01

The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

Discovery of an artificial peptide agonist to the fibroblast growth factor receptor 1c/βKlotho complex from random peptide T7 phage display.

PubMed

Sakamoto, Kotaro; Kawata, Yayoi; Masuda, Yasushi; Umemoto, Tadashi; Ito, Takashi; Asami, Taiji; Takekawa, Shiro; Ohtaki, Tetsuya; Inooka, Hiroshi

2016-11-04

Fibroblast growth factor receptor-1c (FGFR1c)/βKlotho (KLB) complex is a receptor of fibroblast growth factor 21 (FGF21). Pharmacologically, FGF21 shows anti-obesity and anti-diabetic effects upon peripheral administration. Here, we report the development of an artificial peptide agonist to the FGFR1c/KLB heterodimer complex. The peptide, F91-8A07 (LPGRTCREYPDLWWVRCY), was discovered from random peptide T7 phage display and selectively bound to the FGFR1c/KLB complex, but not to FGFR1c and KLB individually. After subsequent peptide dimerization using a short polyethyleneglycol (PEG) linker, the dimeric F91-8A07 peptide showed higher potent agonist activity than that of FGF21 in cultured primary human adipocytes. Moreover, the dimeric peptide led to an expression of the early growth response protein-1 (Egr-1) mRNA in vivo, which is a target gene of FGFR1c. To the best of our knowledge, this is the first report of a FGFR1c/KLB complex-selective artificial peptide agonist. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

MapReduce implementation of a hybrid spectral library-database search method for large-scale peptide identification.

PubMed

Kalyanaraman, Ananth; Cannon, William R; Latt, Benjamin; 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. The source code along with user documentation are available on http://compbio.eecs.wsu.edu/MR-MSPolygraph. ananth@eecs.wsu.edu; william.cannon@pnnl.gov. Supplementary data are available at Bioinformatics online.

Empirical and bioinformatic characterization of buffalo (Bubalus bubalis) colostrum whey peptides & their angiotensin I-converting enzyme inhibition.

PubMed

Ashok, N R; Aparna, H S

2017-08-01

Whey based peptides are well known for their nutritional and multifunctional properties. In this context, whey proteins from buffalo colostrum & milk were digested by in vitro simulation digestion and analyzed by nano-LC-MS/MS. Functional protein association networks, gene annotations and localization of identified proteins were carried out. An ACE inhibitory peptide sorted from the library was custom synthesized and an in vitro ACE assay was performed. The study led to the identification of 74 small peptides which were clustered into 5 gene functional groups and majority of them were secretory proteins. Among the identified peptides, majority of them were found identical to angiotensin I-converting enzyme (ACE) inhibitors, antioxidant, antimicrobial, immunomodulatory and opioidal peptides. An octapeptide (m/z - 902.51, IQKVAGTW) synthesized was found to inhibit ACE with an IC 50 of 300±2µM. The present investigation thus establishes newer vista for food derived peptides having ACE inhibitory potential for nutraceutical or therapeutic applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Assigning statistical significance to proteotypic peptides via database searches

PubMed Central

Alves, Gelio; Ogurtsov, Aleksey Y.; Yu, Yi-Kuo

2011-01-01

Querying MS/MS spectra against a database containing only proteotypic peptides reduces data analysis time due to reduction of database size. Despite the speed advantage, this search strategy is challenged by issues of statistical significance and coverage. The former requires separating systematically significant identifications from less confident identifications, while the latter arises when the underlying peptide is not present, due to single amino acid polymorphisms (SAPs) or post-translational modifications (PTMs), in the proteotypic peptide libraries searched. To address both issues simultaneously, we have extended RAId’s knowledge database to include proteotypic information, utilized RAId’s statistical strategy to assign statistical significance to proteotypic peptides, and modified RAId’s programs to allow for consideration of proteotypic information during database searches. The extended database alleviates the coverage problem since all annotated modifications, even those occurred within proteotypic peptides, may be considered. Taking into account the likelihoods of observation, the statistical strategy of RAId provides accurate E-value assignments regardless whether a candidate peptide is proteotypic or not. The advantage of including proteotypic information is evidenced by its superior retrieval performance when compared to regular database searches. PMID:21055489

The energy landscape of a selective tumor-homing pentapeptide

PubMed Central

Zanuy, David; Flores-Ortega, Alejandra; Casanovas, Jordi; Curco, David; Nussinov, Ruth; Aleman, Carlos

2009-01-01

Recently, a potentially powerful strategy based on the of phage-display libraries has been presented to target tumors via homing peptides attached to nanoparticles. The Cys-Arg-Glu-Lys-Ala (CREKA) peptide sequence has been identified as a tumor-homing peptide that binds to clotted plasmas proteins present in tumor vessels and interstitium. The aim of this work consists of mapping the conformational profile of CREKA to identify the bioactive conformation. For this purpose, a conformational search procedure based on modified Simulated Annealing combined with Molecular Dynamics was applied to three systems that mimic the experimentally used conditions: (i) the free peptide; (ii) the peptide attached to a nanoparticle; and (iii) the peptide inserted in a phage display protein. In addition, the free peptide was simulated in an ionized aqueous solution environment, which mimics the ionic strength of the physiological medium. Accessible minima of all simulated systems reveal a multiple interaction pattern involving the ionized side chains of Arg, Glu and Lys, which induces a β-turn motif in the backbone observed in all simulated CREKA systems. PMID:18588341

PH-sauvagine from the skin secretion of Phyllomedusa hypochondrialis: A novel CRF-like peptide with smooth muscle contraction activity.

PubMed

Zhou, Yu; Shaw, Chris; Chen, Tianbao

2015-09-15

Amphibian skin, and particularly that of south/Central American phyllomedusine frogs, is supposed to be "a huge factory and store house of a variety of active peptides". The 40 amino acid amphibian CRF-like peptide, sauvagine, is a prototype member of a unique family of these Phyllomedusa skin peptides. In this study, we describe for the first time the structure of a mature novel peptide from the skin secretion of the South American orange-legged leaf frog, Phyllomedusa hypochondrialis, which belongs to the amphibian CRF/sauvagine family. Partial amino acid sequence from the N-terminal was obtained by automated Edman degradation with the following structure: pGlu-GPPISIDLNMELLRNMIEI-. The biosynthetic precursor of this novel sauvagine peptide, consisted of 85 amino acid residues and was deduced from cDNA library constructed from the same skin secretion. Compared with the standard sauvagine from the frog, Phyllomedusa sauvagei, this novel peptide was found to exert similar contraction effects on isolated guinea-pig colon and rat urinary bladder smooth muscle preparations. Copyright © 2015 Elsevier Ltd. All rights reserved.

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. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MaRiMba: A Software Application for Spectral Library-Based MRM Transition List Assembly

PubMed Central

Sherwood, Carly A.; Eastham, Ashley; Lee, Lik Wee; Peterson, Amelia; Eng, Jimmy K.; Shteynberg, David; Mendoza, Luis; Deutsch, Eric W.; Risler, Jenni; Tasman, Natalie; Aebersold, Ruedi; Lam, Henry; Martin, Daniel B.

2009-01-01

Multiple reaction monitoring mass spectrometry (MRM-MS) is a targeted analysis method that has been increasingly viewed as an avenue to explore proteomes with unprecedented sensitivity and throughput. We have developed a software tool, called MaRiMba, to automate the creation of explicitly defined MRM transition lists required to program triple quadrupole mass spectrometers in such analyses. MaRiMba creates MRM transition lists from downloaded or custom-built spectral libraries, restricts output to specified proteins or peptides, and filters based on precursor peptide and product ion properties. MaRiMba can also create MRM lists containing corresponding transitions for isotopically heavy peptides, for which the precursor and product ions are adjusted according to user specifications. This open-source application is operated through a graphical user interface incorporated into the Trans-Proteomic Pipeline, and it outputs the final MRM list to a text file for upload to MS instruments. To illustrate the use of MaRiMba, we used the tool to design and execute an MRM-MS experiment in which we targeted the proteins of a well-defined and previously published standard mixture. PMID:19603829

Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

PubMed

Lang, Qiaolin; Wang, Fei; Yin, Long; Liu, Mingjun; Petrenko, Valery A; Liu, Aihua

2014-03-04

Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

Affinity reagent technology development and application to rapid immunochromatographic pathogen detection

NASA Astrophysics Data System (ADS)

Sooter, Letha J.; Stratis-Cullum, Dimitra N.; Zhang, Yanting; Daugherty, Patrick S.; Soh, H. Tom; Pellegrino, Paul; Stagliano, Nancy

2007-09-01

Immunochromatography is a rapid, reliable, and cost effective method of detecting biowarfare agents. The format is similar to that of an over-the-counter pregnancy test. A sample is applied to one end of a cassette and then a control line, and possibly a sample line, are visualized at the other end of the cassette. The test is based upon a sandwich assay. For the control, a line of Protein A is immobilized on the membrane. Gold nanoparticle bound IgG flows through the membrane and binds the Protein A, creating a visible line on the membrane. For the sample, one epitope is immobilized on the membrane and another epitope is attached to gold nanoparticles. The sample binds gold bound epitope, travels through the membrane, and binds membrane bound epitope. The two epitopes are not cross-reactive, therefore a sample line is only visible if the sample is present. In order to efficiently screen for binders to a sample target, a novel, Continuous Magnetic Activated Cell Sorter (CMACS) has been developed on a disposable, microfluidic platform. The CMACS chip quickly sorts E. coli peptide libraries for target binders with high affinity. Peptide libraries, are composed of approximately ten million bacteria, each displaying a different peptide on their surface. The target of interest is conjugated to a micrometer sized magnetic particle. After the library and the target are incubated together to allow binding, the mixture is applied to the CMACS chip. In the presence of patterned nickel and an external magnet, separation occurs of the bead-bound bacteria from the bulk material. The bead fraction is added to bacterial growth media where any attached E. coli grow and divide. These cells are cloned, sequenced, and the peptides are assayed for target binding affinity. As a proof-of-principle, assays were developed for human C-reactive protein. More defense relevant targets are currently being pursued.

Meta sequence analysis of human blood peptides and their parent proteins.

PubMed

Bowden, Peter; Pendrak, Voitek; Zhu, Peihong; Marshall, John G

2010-04-18

Sequence analysis of the blood peptides and their qualities will be key to understanding the mechanisms that contribute to error in LC-ESI-MS/MS. Analysis of peptides and their proteins at the level of sequences is much more direct and informative than the comparison of disparate accession numbers. A portable database of all blood peptide and protein sequences with descriptor fields and gene ontology terms might be useful for designing immunological or MRM assays from human blood. The results of twelve studies of human blood peptides and/or proteins identified by LC-MS/MS and correlated against a disparate array of genetic libraries were parsed and matched to proteins from the human ENSEMBL, SwissProt and RefSeq databases by SQL. The reported peptide and protein sequences were organized into an SQL database with full protein sequences and up to five unique peptides in order of prevalence along with the peptide count for each protein. Structured query language or BLAST was used to acquire descriptive information in current databases. Sampling error at the level of peptides is the largest source of disparity between groups. Chi Square analysis of peptide to protein distributions confirmed the significant agreement between groups on identified proteins. Copyright 2010. Published by Elsevier B.V.

Effects of 8-mer acidic peptide concentration on the morphology and photoluminescence of synthesized ZnO nanomaterials

NASA Astrophysics Data System (ADS)

Moon, Chung Hee; Tousi, Marzieh; Cheeney, Joseph; Ngo-Duc, Tam-Triet; Zuo, Zheng; Liu, Jianlin; Haberer, Elaine D.

2015-11-01

An 8-mer ZnO-binding peptide, VPGAAEHT, was identified using a M13 pVIII phage display library and employed as an additive during aqueous-based ZnO synthesis at 65 °C. Unlike most other well-studied ZnO-binding sequences which are strongly basic (pI > pH 7), the 8-mer peptide was overall acidic (pI < pH 7) in character, including only a single basic residue. The selected peptide strongly influenced ZnO nanostructure formation. Morphology and optical emission properties were found to be dependent on the concentration of peptide additive. Using lower peptide concentrations (<0.1 mM), single crystal hexagonal rods and platelets were produced, and using higher peptide concentrations (≥0.1 mM), polycrystalline layered platelets, yarn-like structures, and microspheres were assembled. Photoluminescence analysis revealed a characteristic ZnO band-edge peak, as well as sub-bandgap emission peaks. Defect-related green emission, typically associated with surface-related oxygen and zinc vacancies, was significantly reduced by the peptide additive, while blue emission, attributable to oxygen and zinc interstitials, emerged with increased peptide concentrations. Peptide-directed synthesis of ZnO materials may be useful for gas sensing and photocatalytic applications in which properly engineered morphology and defect levels have demonstrated enhanced performance.

SIMULATION AND VISUALIZATION OF FLOW PATTERN IN MICROARRAYS FOR LIQUID PHASE OLIGONUCLEOTIDE AND PEPTIDE SYNTHESIS

PubMed Central

O-Charoen, Sirimon; Srivannavit, Onnop; Gulari, Erdogan

2008-01-01

Microfluidic microarrays have been developed for economical and rapid parallel synthesis of oligonucleotide and peptide libraries. For a synthesis system to be reproducible and uniform, it is crucial to have a uniform reagent delivery throughout the system. Computational fluid dynamics (CFD) is used to model and simulate the microfluidic microarrays to study geometrical effects on flow patterns. By proper design geometry, flow uniformity could be obtained in every microreactor in the microarrays. PMID:17480053

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…

Quantitative measurement of phosphoproteome response to osmotic stress in arabidopsis based on Library-Assisted eXtracted Ion Chromatogram (LAXIC).

PubMed

Xue, Liang; Wang, Pengcheng; Wang, Lianshui; Renzi, Emily; Radivojac, Predrag; Tang, Haixu; Arnold, Randy; Zhu, Jian-Kang; Tao, W Andy

2013-08-01

Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants.

MzJava: An open source library for mass spectrometry data processing.

PubMed

Horlacher, Oliver; Nikitin, Frederic; Alocci, Davide; Mariethoz, Julien; Müller, Markus; Lisacek, Frederique

2015-11-03

Mass spectrometry (MS) is a widely used and evolving technique for the high-throughput identification of molecules in biological samples. The need for sharing and reuse of code among bioinformaticians working with MS data prompted the design and implementation of MzJava, an open-source Java Application Programming Interface (API) for MS related data processing. MzJava provides data structures and algorithms for representing and processing mass spectra and their associated biological molecules, such as metabolites, glycans and peptides. MzJava includes functionality to perform mass calculation, peak processing (e.g. centroiding, filtering, transforming), spectrum alignment and clustering, protein digestion, fragmentation of peptides and glycans as well as scoring functions for spectrum-spectrum and peptide/glycan-spectrum matches. For data import and export MzJava implements readers and writers for commonly used data formats. For many classes support for the Hadoop MapReduce (hadoop.apache.org) and Apache Spark (spark.apache.org) frameworks for cluster computing was implemented. The library has been developed applying best practices of software engineering. To ensure that MzJava contains code that is correct and easy to use the library's API was carefully designed and thoroughly tested. MzJava is an open-source project distributed under the AGPL v3.0 licence. MzJava requires Java 1.7 or higher. Binaries, source code and documentation can be downloaded from http://mzjava.expasy.org and https://bitbucket.org/sib-pig/mzjava. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Biomolecule mediating synthesis of inorganic nanoparticles and their applications

NASA Astrophysics Data System (ADS)

Wei, Zengyan

Project 1. The conventional phage display technique focuses on screening peptide sequences that can bind on target substrates, however the selected peptides are not necessary to nucleate and mediate the growth of the target inorganic crystals, and in many cases they only show moderate affinity to the targets. Here we report a novel phage display approach that can directly screen peptides catalytically growing inorganic nanoparticles in aqueous solution at room temperature. In this study, the phage library is incubated with zinc precursor at room temperature. Among random peptide sequences displayed on phages, those phages that can grow zinc oxide (ZnO) nanoparticles are selected with centrifugation. After several rounds of selection, the peptide sequences displayed on the phage viruses are analyzed by DNA sequencing. Our screening protocol provide a simple and convenient route for the discovery of catalytic peptides that can grow inorganic nanoparticles at room temperature. This novel screening protocol can extend the method on finding a wide range of new catalysts. Project 2. Genetically engineered collagen peptides are assembled into freestanding films when quantum dots (QDs) are co-assembled as joints between collagen domains. These peptide-based films show excellent mechanical properties with Young's modulus of 20 GPa, much larger than most of the multi-composite polymer films and previously reported freestanding nanoparticle-assembled sheets, and it is even close to that reported for the bone tissue in nature. These films show little permanent deformation under small indentation while the mechanical hysteresis becomes remarkable when the load approaches near and beyond the rupture point, which is also characteristic of the bone tissue. Project 3. The shape-controlled synthesis of nanoparticles have been established in single-phase solutions by controlling growth directions of crystalline facets on seed nanocrystals kinetically; however, it is difficult to rationally predict and design nanoparticle shapes. Here we introduce a methodology to fabricate nanoparticles in smaller sizes by evolving shapes thermodynamically. This strategy enables a more rational approach to fabricate shaped nanoparticles by etching specific positions of atoms on facets of seed nanocrystals in reverse micelle reactors where the surface energy gradient induces desorption of atoms on specific locations on the seed surfaces. From seeds of 12 nm palladium nanocubes, the shape is evolved to concave nanocubes and finally hollow nanocages in the size 10 nm by etching the center of {200} facets. The high surface area-to-volume ratio and the exposure of a large number of palladium atoms on ledge and kink sites of hollow nanocages are advantageous to enhance catalytic activity and recyclability.

Unifying expression scale for peptide hydrophobicity in proteomic reversed phase high-pressure liquid chromatography experiments.

PubMed

Grigoryan, Marine; Shamshurin, Dmitry; Spicer, Victor; Krokhin, Oleg V

2013-11-19

As an initial step in our efforts to unify the expression of peptide retention times in proteomic liquid chromatography-mass spectrometry (LC-MS) experiments, we aligned the chromatographic properties of a number of peptide retention standards against a collection of peptides commonly observed in proteomic experiments. The standard peptide mixtures and tryptic digests of samples of different origins were separated under the identical chromatographic condition most commonly employed in proteomics: 100 Å C18 sorbent with 0.1% formic acid as an ion-pairing modifier. Following our original approach (Krokhin, O. V.; Spicer, V. Anal. Chem. 2009, 81, 9522-9530) the retention characteristics of these standards and collection of tryptic peptides were mapped into hydrophobicity index (HI) or acetonitrile percentage units. This scale allows for direct visualization of the chromatographic outcome of LC-MS acquisitions, monitors the performance of the gradient LC system, and simplifies method development and interlaboratory data alignment. Wide adoption of this approach would significantly aid understanding the basic principles of gradient peptide RP-HPLC and solidify our collective efforts in acquiring confident peptide retention libraries, a key component in the development of targeted proteomic approaches.

PROCAL: A Set of 40 Peptide Standards for Retention Time Indexing, Column Performance Monitoring, and Collision Energy Calibration.

PubMed

Zolg, Daniel Paul; Wilhelm, Mathias; Yu, Peng; Knaute, Tobias; Zerweck, Johannes; Wenschuh, Holger; Reimer, Ulf; Schnatbaum, Karsten; Kuster, Bernhard

2017-11-01

Beyond specific applications, such as the relative or absolute quantification of peptides in targeted proteomic experiments, synthetic spike-in peptides are not yet systematically used as internal standards in bottom-up proteomics. A number of retention time standards have been reported that enable chromatographic aligning of multiple LC-MS/MS experiments. However, only few peptides are typically included in such sets limiting the analytical parameters that can be monitored. Here, we describe PROCAL (ProteomeTools Calibration Standard), a set of 40 synthetic peptides that span the entire hydrophobicity range of tryptic digests, enabling not only accurate determination of retention time indices but also monitoring of chromatographic separation performance over time. The fragmentation characteristics of the peptides can also be used to calibrate and compare collision energies between mass spectrometers. The sequences of all selected peptides do not occur in any natural protein, thus eliminating the need for stable isotope labeling. We anticipate that this set of peptides will be useful for multiple purposes in individual laboratories but also aiding the transfer of data acquisition and analysis methods between laboratories, notably the use of spectral libraries. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bio-inspired synthetic receptor molecules towards mimicry of vancomycin.

PubMed

Monnee, M C; Brouwer, A J; Verbeek, L M; van Wageningen, A M; Liskamp, R M

2001-06-18

A 512-member library of bio-inspired synthetic receptor molecules was prepared featuring a triazacyclophane scaffold. The purpose of this scaffold was to orient three (identical) peptide 'binding arms' in order to mimic an antibiotic binding cavity as is present in the vancomycin antibiotics. The library was screened with D-Ala-D-Ala and D-Ala-D-Lac containing ligands, which are present in the cell wall precursors of pathogenic bacteria. Screening and validation led to identification of a synthetic receptor capable of binding these ligands.

Biomagnetic separation of Salmonella Typhimurium with high affine and specific ligand peptides isolated by phage display technique

NASA Astrophysics Data System (ADS)

Steingroewer, Juliane; Bley, Thomas; Bergemann, Christian; Boschke, Elke

2007-04-01

Analyses of food-borne pathogens are of great importance in order to minimize the health risk for customers. Thus, very sensitive and rapid detection methods are required. Current conventional culture techniques are very time consuming. Modern immunoassays and biochemical analysis also require pre-enrichment steps resulting in a turnaround time of at least 24 h. Biomagnetic separation (BMS) is a promising more rapid method. In this study we describe the isolation of high affine and specific peptides from a phage-peptide library, which combined with BMS allows the detection of Salmonella spp. with a similar sensitivity as that of immunomagnetic separation using antibodies.

PERMutation Using Transposase Engineering (PERMUTE): A Simple Approach for Constructing Circularly Permuted Protein Libraries.

PubMed

Jones, Alicia M; Atkinson, Joshua T; Silberg, Jonathan J

2017-01-01

Rearrangements that alter the order of a protein's sequence are used in the lab to study protein folding, improve activity, and build molecular switches. One of the simplest ways to rearrange a protein sequence is through random circular permutation, where native protein termini are linked together and new termini are created elsewhere through random backbone fission. Transposase mutagenesis has emerged as a simple way to generate libraries encoding different circularly permuted variants of proteins. With this approach, a synthetic transposon (called a permuteposon) is randomly inserted throughout a circularized gene to generate vectors that express different permuted variants of a protein. In this chapter, we outline the protocol for constructing combinatorial libraries of circularly permuted proteins using transposase mutagenesis, and we describe the different permuteposons that have been developed to facilitate library construction.

Mapping of binding epitopes of a human decay-accelerating factor monoclonal antibody capable of enhancing rituximab-mediated complement-dependent cytotoxicity.

PubMed

Guo, Bo; Ma, Zheng-wei; Li, Hua; Xu, Gui-lian; Zheng, Ping; Zhu, Bo; Wu, Yu-Zhang; Zou, Qiang

2008-08-01

Complement-dependent cytotoxicity (CDC) is thought to be one of the most important mechanisms of action of therapeutic monoclonal antibodies (mAbs). The decay-accelerating factor (DAF) overexpressed in certain tumors limits the CDC effect of the therapeutic anticancer antibodies. The use of DAF blocking antibodies targeted specifically at cancer cells in combination with immunotherapeutic mAbs of cancer may improve the therapeutic effect in cancer patients. In this study, the lysis of Raji cells mediated by CDC was determined after blocking DAF function by anti-DAF polyclonal antibody and 3 mAbs (DG3, DG9, DA11) prepared in our laboratory, respectively, in the presence of the anti-CD20 chimeric mAb rituximab. The binding domains of the three anti-DAF mAbs were identified using yeast surface display technique, and the mimic epitopes of mAb DG3 were screened from a random phage-display nonapeptide library. The results showed that blocking DAF function by anti-DAF polyclonal antibody enhanced complement-mediated killing of Raji cells. Among the 3 mAbs against DAF, only DG3 was found to be able to remarkably enhance the CDC effect of the therapeutic mAb rituximab. DG3 bound to the third short consensus repeat (SCR) of DAF. Binding of DG3 to immobilized DAF was inhibited by mimic epitope peptides screened from the peptide library. Our results suggest that a higher level of DAF expressed by certain tumor cells is significant to abolish the CDC effect of therapeutic anticancer antibodies, and mAbs binding to SCR3 can enhance the complement-mediated killing of Raji cells. It is of significance to identify the DAF epitopes required in inhibiting CDC not only for better understanding of the relationship between the structure and function of DAF, but also for designing and developing anti-DAF mAbs capable of enhancing CDC.

An extensive library of surrogate peptides for all human proteins.

PubMed

Mohammed, Yassene; Borchers, Christoph H

2015-11-03

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Discovery of a polystyrene binding peptide isolated from phage display library and its application in peptide immobilization.

PubMed

Qiang, Xu; Sun, Keyong; Xing, Lijun; Xu, Yifeng; Wang, Hong; Zhou, Zhengpin; Zhang, Juan; Zhang, Fang; Caliskan, Bilgen; Wang, Min; Qiu, Zheng

2017-06-01

Phage peptide display is a powerful technique for discovery of various target-specific ligands. However, target-unrelated peptides can often be obtained and cause ambiguous results. Peptide PB-TUP has been isolated repeatedly in our laboratory on different targets and we conducted a research on PB-TUP phage to investigate their binding properties and rate of propagation. ELISA and phage recovery assay demonstrated that PB-TUP phage had a significant superior affinity to polystyrene solid surface compared with control phage clones. In this study, some incidental bindings are excluded like blocking agents and non-specific binding of secondary antibodies. Propagation rate assays of the selected phage clones showed that the growth rate of PB-TUP phage was not superior to the control phages. Furthermore, the binding of PB-TUB to polystyrene was concentration dependent and varied with solution pH. Molecular modeling revealed that stable structures of α-helix and β-turn may contribute to the binding of PB-TUP to polystyrene plate. The PB-TUP sequence was fused to the N-terminus of peptide P2 and the fusion peptide significantly increased the binding affinity to polystyrene. The fusion peptide also enhanced the cell adhesion ability of peptide P2 with human umbilical vein endothelial cell (HUVEC). The addition of the polystyrene binding peptide provided a convenient method for peptide immobilization.

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

PubMed

Zhao, Jing; Frauenkron-Machedjou, Victorine Josiane; Kardashliev, Tsvetan; Ruff, Anna Joëlle; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich

2017-04-01

The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.

Organic crystal-binding peptides: morphology control and one-pot formation of protein-displaying organic crystals

NASA Astrophysics Data System (ADS)

Niide, Teppei; Ozawa, Kyohei; Nakazawa, Hikaru; Oliveira, Daniel; Kasai, Hitoshi; Onodera, Mari; Asano, Ryutaro; Kumagai, Izumi; Umetsu, Mitsuo

2015-11-01

Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals. Electronic supplementary information (ESI) available: Schematic representation of PeryBPb1-fused DsRed-Monomer, fluorescence spectra of perylene crystals and DsRed-Monomer, and emission spectra of DsRed-Monomer at various excitation wavelengths. See DOI: 10.1039/c5nr06471f

Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

NASA Astrophysics Data System (ADS)

Essler, Markus; Ruoslahti, Erkki

2002-02-01

In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.

Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Checco, James W.; Kreitler, Dale F.; Thomas, Nicole C.

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. In this paper, we describe a strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF 165-induced proliferation of human umbilical vein endothelialmore » cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Finally, such reagents would be useful for diagnostic and therapeutic applications.« less

Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

DOE PAGES

Checco, James W.; Kreitler, Dale F.; Thomas, Nicole C.; ...

2015-03-30

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. In this paper, we describe a strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF 165-induced proliferation of human umbilical vein endothelialmore » cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Finally, such reagents would be useful for diagnostic and therapeutic applications.« less

Crossing borders to bind proteins--a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set.

PubMed

Baltzer, Lars

2011-06-01

A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Xiaoyong; Cai, Cuizan; Xiao, Fei

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 havemore » 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.« less

Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection

NASA Astrophysics Data System (ADS)

Tsao, Shih-Ming; Lai, Ji-Ching; Horng, Horng-Er; Liu, Tu-Chen; Hong, Chin-Yih

2017-04-01

Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5‧ and 3‧ termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

Purification, characterization and molecular cloning of chymotrypsin inhibitor peptides from the venom of Burmese Daboia russelii siamensis.

PubMed

Guo, Chun-Teng; McClean, Stephen; Shaw, Chris; Rao, Ping-Fan; Ye, Ming-Yu; Bjourson, Anthony J

2013-05-01

One novel Kunitz BPTI-like peptide designated as BBPTI-1, with chymotrypsin inhibitory activity was identified from the venom of Burmese Daboia russelii siamensis. It was purified by three steps of chromatography including gel filtration, cation exchange and reversed phase. A partial N-terminal sequence of BBPTI-1, HDRPKFCYLPADPGECLAHMRSF was obtained by automated Edman degradation and a Ki value of 4.77nM determined. Cloning of BBPTI-1 including the open reading frame and 3' untranslated region was achieved from cDNA libraries derived from lyophilized venom using a 3' RACE strategy. In addition a cDNA sequence, designated as BBPTI-5, was also obtained. Alignment of cDNA sequences showed that BBPTI-5 exhibited an identical sequence to BBPTI-1 cDNA except for an eight nucleotide deletion in the open reading frame. Gene variations that represented deletions in the BBPTI-5 cDNA resulted in a novel protease inhibitor analog. Amino acid sequence alignment revealed that deduced peptides derived from cloning of their respective precursor cDNAs from libraries showed high similarity and homology with other Kunitz BPTI proteinase inhibitors. BBPTI-1 and BBPTI-5 consist of 60 and 66 amino acid residues respectively, including six conserved cysteine residues. As these peptides have been reported to have influence on the processes of coagulation, fibrinolysis and inflammation, their potential application in biomedical contexts warrants further investigation. Copyright © 2013 Elsevier Inc. All rights reserved.

A Diversity-Oriented Library of Fluorophore-Modified Receptors Constructed from a Chemical Library of Synthetic Fluorophores.

PubMed

Nakano, Shun; Tamura, Tomoki; Das, Raj Kumar; Nakata, Eiji; Chang, Young-Tae; Morii, Takashi

2017-11-16

The practical application of biosensors can be determined by evaluating the sensing ability of fluorophore-modified derivatives of a receptor with appropriate recognition characteristics for target molecules. One of the key determinants for successfully obtaining a useful biosensor is wide variation in the fluorophores attached to a given receptor. Thus, using a larger fluorophore-modified receptor library provides a higher probability of obtaining a practically useful biosensor. However, no effective method has yet been developed for constructing such a diverse library of fluorophore-modified receptors. Herein, we report a method for constructing fluorophore-modified receptors by using a chemical library of synthetic fluorophores with a thiol-reactive group. This library was converted into a library of fluorophore-modified adenosine-binding ribonucleopeptide (RNP) receptors by introducing the fluorophores to the Rev peptide of the RNP complex by alkylation of the thiol group. This method enabled the construction of 263 fluorophore-modified ATP-binding RNP receptors and allowed the selection of suitable receptor-based fluorescent sensors that target ATP. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modification Site Localization in Peptides.

PubMed

Chalkley, Robert J

2016-01-01

There are a large number of search engines designed to take mass spectrometry fragmentation spectra and match them to peptides from proteins in a database. These peptides could be unmodified, but they could also bear modifications that were added biologically or during sample preparation. As a measure of reliability for the peptide identification, software normally calculates how likely a given quality of match could have been achieved at random, most commonly through the use of target-decoy database searching (Elias and Gygi, Nat Methods 4(3): 207-214, 2007). Matching the correct peptide but with the wrong modification localization is not a random match, so results with this error will normally still be assessed as reliable identifications by the search engine. Hence, an extra step is required to determine site localization reliability, and the software approaches to measure this are the subject of this part of the chapter.

One-step random mutagenesis by error-prone rolling circle amplification

PubMed Central

Fujii, Ryota; Kitaoka, Motomitsu; Hayashi, Kiyoshi

2004-01-01

In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3–4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique. PMID:15507684

Cationic antimicrobial peptides inactivate Shiga toxin-encoding bacteriophages

NASA Astrophysics Data System (ADS)

Del Cogliano, Manuel E.; Hollmann, Axel; Martinez, Melina; Semorile, Liliana; Ghiringhelli, Pablo D.; Maffía, Paulo C.; Bentancor, Leticia V.

2017-12-01

Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: 1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, 2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and 3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

Buwchitin: a ruminal peptide with antimicrobial potential against Enterococcus faecalis

NASA Astrophysics Data System (ADS)

Oyama, Linda B.; Crochet, Jean-Adrien; Edwards, Joan E.; Girdwood, Susan E.; Cookson, Alan R.; Fernandez-Fuentes, Narcis; Hilpert, Kai; Golyshin, Peter N.; Golyshina, Olga V.; Privé, Florence; Hess, Matthias; Mantovani, Hilario C.; Creevey, Christopher J.; Huws, Sharon A.

2017-07-01

Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

A phage display-selected peptide inhibitor of Agrobacterium vitis polygalacturonase.

PubMed

Warren, Jeremy G; Kasun, George W; Leonard, Takara; Kirkpatrick, Bruce C

2016-05-01

Agrobacterium vitis, the causal agent of crown gall of grapevine, is a threat to viticulture worldwide. A major virulence factor of this pathogen is polygalacturonase, an enzyme that degrades pectin components of the xylem cell wall. A single gene encodes for the polygalacturonase gene. Disruption of the polygalacturonase gene results in a mutant that is less pathogenic and produces significantly fewer root lesions on grapevines. Thus, the identification of peptides or proteins that could inhibit the activity of polygalacturonase could be part of a strategy for the protection of plants against this pathogen. A phage-displayed combinatorial peptide library was used to isolate peptides with a high binding affinity to A. vitis polygalacturonase. These peptides showed sequence similarity to regions of Oryza sativa (EMS66324, Japonica) and Triticum urartu (NP_001054402, wild wheat) polygalacturonase-inhibiting proteins (PGIPs). Furthermore, these panning experiments identified a peptide, SVTIHHLGGGS, which was able to reduce A. vitis polygalacturonase activity by 35% in vitro. Truncation studies showed that the IHHL motif alone is sufficient to inhibit A. vitis polygalacturonase activity. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Comprehensive computational design of ordered peptide macrocycles

PubMed Central

Hosseinzadeh, Parisa; Bhardwaj, Gaurav; Mulligan, Vikram Khipple; Shortridge, Matthew D.; Craven, Timothy W.; Pardo-Avila, Fátima; Rettie, Stephen A.; Kim, David E.; Silva, Daniel-Adriano; Ibrahim, Yehia M.; Webb, Ian K.; Cort, John R.; Adkins, Joshua N.; Varani, Gabriele; Baker, David

2018-01-01

Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods. PMID:29242347

A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity.

PubMed

Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min

2015-08-01

Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.

Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein Quantification.

PubMed

Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Paik, Young-Ki

2017-12-01

Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.

Kinetics of Contact Formation and End-to-End Distance Distributions of Swollen Disordered Peptides

PubMed Central

Soranno, Andrea; Longhi, Renato; Bellini, Tommaso; Buscaglia, Marco

2009-01-01

Unstructured polypeptide chains are subject to various degrees of swelling or compaction depending on the combination of solvent condition and amino acid sequence. Highly denatured proteins generally behave like random-coils with excluded volume repulsion, whereas in aqueous buffer more compact conformations have been observed for the low-populated unfolded state of globular proteins as well as for naturally disordered sequences. To quantitatively account for the different mechanisms inducing the swelling of polypeptides, we have examined three 14-residues peptides in aqueous buffer and in denaturant solutions, including the well characterized AGQ repeat as a reference and two variants, in which we have successively introduced charged side chains and removed the glycines. Quenching of the triplet state of tryptophan by close contact with cysteine has been used in conjunction with Förster resonance energy transfer to study the equilibrium and kinetic properties of the peptide chains. The experiments enable accessing end-to-end root mean-square distance, probability of end-to-end contact formation and intrachain diffusion coefficient. The data can be coherently interpreted on the basis of a simple chain model with backbone angles obtained from a library of coil segments of proteins and hard sphere repulsion at each Cα position. In buffered water, we find that introducing charges in a glycine-rich sequence induces a mild chain swelling and a significant speed-up of the intrachain dynamics, whereas the removal of the glycines results in almost a two-fold increase of the chain volume and a drastic slowing down. In denaturants we observe a pronounced swelling of all the chains, with significant differences between the effect of urea and guanidinium chloride. PMID:19217868

Cancer-associated CD43 glycoforms as target of immunotherapy

PubMed Central

Tuccillo, Franca Maria; Palmieri, Camillo; Fiume, Giuseppe; de Laurentiis, Annamaria; Schiavone, Marco; Falcone, Cristina; Iaccino, Enrico; Galandrini, Ricciarda; Capuano, Cristina; Santoni, Angela; D'Armiento, Francesco Paolo; Arra, Claudio; Barbieri, Antonio; Piaz, Fabrizio Dal; Venzon, David; Bonelli, Patrizia; Buonaguro, Franco Maria; Scala, Iris; Mallardo, Massimo; Quinto, Ileana; Scala, Giuseppe

2014-01-01

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer–association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with anti-tumor activity in vivo since its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T-cells in mice. Further, we demonstrate that tumor inhibition was due to UN1 mAb-dependent NK-mediated cytotoxicity. By screening a phage displayed random peptide library we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harboured a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumour cells. Based on sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility to use monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy. PMID:24356816

Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

PubMed

Kulabhusan, Prabir Kumar; Rajwade, Jyutika M; Sahul Hameed, A S; Paknikar, Kishore M

2017-06-01

White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (K d ,app) was calculated to be 810 nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5 μg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20 min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

PubMed Central

Subramanian, Shoba; Hardt, Markus; Choe, Youngchool; Niles, Richard K.; Johansen, Eric B.; Legac, Jennifer; Gut, Jiri; Kerr, Iain D.; Craik, Charles S.; Rosenthal, Philip J.

2009-01-01

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P1 – P4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P2 position. Second, with overlapping peptides spanning α and β globin and proteolysis-dependent 18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents. PMID:19357776

Identification of a novel autoantibody against self-vimentin specific in secondary Sjögren's syndrome.

PubMed

Li, Yu-Hui; Gao, Ya-Ping; Dong, Jie; Shi, Lian-Jie; Sun, Xiao-Lin; Li, Ru; Zhang, Xue-Wu; Liu, Yu; Long, Li; He, Jing; Zhong, Qun-Jie; Morand, Eric; Yang, Guang; Li, Zhan-Guo

2018-02-12

Sjögren's syndrome (SS) is a primary autoimmune disease (pSS) or secondarily associated with other autoimmune diseases (sSS). The mechanisms underlying immune dysregulation in this syndrome remain unknown, and clinically it is difficult to diagnose owing to a lack of specific biomarkers. We extracted immunoglobulins (Igs) from the sera of patients with sSS associated with rheumatoid arthritis (RA) and used them to screen a phage display library of peptides with random sequences. Our results show that an sSS-specific peptide, designated 3S-P, was recognized by sera of 68.2% (60 of 88) patients with sSS, 66.2% of patients with RA-sSS, and 76.5% of patients with systemic lupus erythematosus (SLE)-sSS. The anti-3S-P antibody was scarcely found in patients with pSS (1.8%), RA (1.3%), SLE (4.2%), ankylosing spondylitis (0%), and gout (3.3%), as well as in healthy donors (2%). The 3S-P-binding Igs (antibodies) were used to identify antigens from salivary glands and synovial tissues from patients with sSS. A putative target autoantigen expressed in the synovium and salivary gland recognized by anti-3S-P antibody was identified as self-vimentin. This novel autoantibody is highly specific in the diagnosis of sSS, and the underlying molecular mechanism of the disease might be epitope spreading involved with vimentin.

Selection of phage-displayed peptides for the detection of imidacloprid in water and soil.

PubMed

Liu, Zhiping; Liu, Jianfeng; Wang, Kai; Li, Wenhui; Shelver, Weilin L; Li, Qing X; Li, Ji; Xu, Ting

2015-09-15

Imidacloprid is the most widely used neonicotinoid insecticide in the world and shows widespread environment and human exposures. A phage clone designated L7-1 that selectively binds to imidacloprid was selected from a commercial phage display library containing linear 7-mer randomized amino acid residues. Using the clone L7-1, a competitive enzyme-linked immunosorbent assay (ELISA) for imidacloprid was developed. The half-maximum signal inhibition concentration (IC50) and the limit of detection (LOD) of the phage ELISA for imidacloprid were 96 and 2.3 ng ml(-1), respectively. This phage ELISA showed relatively low cross-reactivity with all of the tested compounds structurally similar to imidacloprid, less than 2% with the exception of 6-chloronicotinic acid, a metabolite of imidacloprid that showed 11.5%. The average recoveries of the phage ELISA for imidacloprid in water and soil samples were in the ranges of 74.6 to 86.3% and 72.5 to 93.6%, respectively. The results of the competitive phage ELISA for imidacloprid in the fortified samples agreed well with those of a high-performance liquid chromatography (HPLC) method. The simple phage-displayed peptide technology has been proven to be a convenient and efficient method for the development of an alternative format of ELISA for small molecules. Copyright © 2015 Elsevier Inc. All rights reserved.

Rosetta FlexPepDock ab-initio: simultaneous folding, docking and refinement of peptides onto their receptors.

PubMed

Raveh, Barak; London, Nir; Zimmerman, Lior; Schueler-Furman, Ora

2011-04-29

Flexible peptides that fold upon binding to another protein molecule mediate a large number of regulatory interactions in the living cell and may provide highly specific recognition modules. We present Rosetta FlexPepDock ab-initio, a protocol for simultaneous docking and de-novo folding of peptides, starting from an approximate specification of the peptide binding site. Using the Rosetta fragments library and a coarse-grained structural representation of the peptide and the receptor, FlexPepDock ab-initio samples efficiently and simultaneously the space of possible peptide backbone conformations and rigid-body orientations over the receptor surface of a given binding site. The subsequent all-atom refinement of the coarse-grained models includes full side-chain modeling of both the receptor and the peptide, resulting in high-resolution models in which key side-chain interactions are recapitulated. The protocol was applied to a benchmark in which peptides were modeled over receptors in either their bound backbone conformations or in their free, unbound form. Near-native peptide conformations were identified in 18/26 of the bound cases and 7/14 of the unbound cases. The protocol performs well on peptides from various classes of secondary structures, including coiled peptides with unusual turns and kinks. The results presented here significantly extend the scope of state-of-the-art methods for high-resolution peptide modeling, which can now be applied to a wide variety of peptide-protein interactions where no prior information about the peptide backbone conformation is available, enabling detailed structure-based studies and manipulation of those interactions. © 2011 Raveh et al.

Rosetta FlexPepDock ab-initio: Simultaneous Folding, Docking and Refinement of Peptides onto Their Receptors

PubMed Central

Raveh, Barak; London, Nir; Zimmerman, Lior; Schueler-Furman, Ora

2011-01-01

Flexible peptides that fold upon binding to another protein molecule mediate a large number of regulatory interactions in the living cell and may provide highly specific recognition modules. We present Rosetta FlexPepDock ab-initio, a protocol for simultaneous docking and de-novo folding of peptides, starting from an approximate specification of the peptide binding site. Using the Rosetta fragments library and a coarse-grained structural representation of the peptide and the receptor, FlexPepDock ab-initio samples efficiently and simultaneously the space of possible peptide backbone conformations and rigid-body orientations over the receptor surface of a given binding site. The subsequent all-atom refinement of the coarse-grained models includes full side-chain modeling of both the receptor and the peptide, resulting in high-resolution models in which key side-chain interactions are recapitulated. The protocol was applied to a benchmark in which peptides were modeled over receptors in either their bound backbone conformations or in their free, unbound form. Near-native peptide conformations were identified in 18/26 of the bound cases and 7/14 of the unbound cases. The protocol performs well on peptides from various classes of secondary structures, including coiled peptides with unusual turns and kinks. The results presented here significantly extend the scope of state-of-the-art methods for high-resolution peptide modeling, which can now be applied to a wide variety of peptide-protein interactions where no prior information about the peptide backbone conformation is available, enabling detailed structure-based studies and manipulation of those interactions. PMID:21572516

Targeting the prostate for destruction through a vascular address

PubMed Central

Arap, Wadih; Haedicke, Wolfgang; Bernasconi, Michele; Kain, Renate; Rajotte, Daniel; Krajewski, Stanislaw; Ellerby, H. Michael; Bredesen, Dale E.; Pasqualini, Renata; Ruoslahti, Erkki

2002-01-01

Organ specific drug targeting was explored in mice as a possible alternative to surgery to treat prostate diseases. Peptides that specifically recognize the vasculature in the prostate were identified from phage-displayed peptide libraries by selecting for phage capable of homing into the prostate after an i.v. injection. One of the phage selected in this manner homed to the prostate 10–15 times more than to other organs. Unselected phage did not show this preference. The phage bound also to vasculature in the human prostate. The peptide displayed by the prostate-homing phage, SMSIARL (single letter code), was synthesized and shown to inhibit the homing of the phage when co-injected into mice with the phage. Systemic treatment of mice with a chimeric peptide consisting of the SMSIARL homing peptide, linked to a proapoptotic peptide that disrupts mitochondrial membranes, caused tissue destruction in the prostate, but not in other organs. The chimeric peptide delayed the development of the cancers in prostate cancer-prone transgenic mice (TRAMP mice). These results suggest that it may be possible to develop an alternative to surgical prostate resection and that such a treatment may also reduce future cancer risk. PMID:11830668

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinitymore » to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 {+-} 0.7 x 10{sup 5} M{sup -1} which indicates a strong binding close to that of antibody.« less

Peptides selected for the protein nanocage pores change the rate of iron recovery from the ferritin mineral.

PubMed

Liu, Xiaofeng S; Patterson, Leslie D; Miller, Marvin J; Theil, Elizabeth C

2007-11-02

Pores regulate access between ferric-oxy biomineral inside and reductants/chelators outside the ferritin protein nanocage to control iron demineralization rates. The pore helix/loop/helix motifs that are contributed by three subunits unfold independently of the protein cage, as observed by crystallography, Fe removal rates, and CD spectroscopy. Pore unfolding is induced in wild type ferritin by increased temperature or urea (1-10 mM), a physiological urea range, 0.1 mM guanidine, or mutation of conserved pore amino acids. A peptide selected for ferritin pore binding from a combinatorial, heptapeptide library increased the rate of Fe demineralization 3-fold (p<0.001), similarly to a mutation that unfolded the pores. Conjugating the peptide to Desferal (desferrioxamine B mesylate), a chelator in therapeutic use, increased the rates to 8-fold (p<0.001). A second pore binding peptide had the opposite effect and decreased the rate of Fe demineralization 60% (p<0.001). The peptides could have pharmacological uses and may model regulators of ferritin demineralization rates in vivo or peptide regulators of gated pores in membranes. The results emphasize that small peptides can exploit the structural plasticity of protein pores to modulate function.

Tailored Synthesis of 162-Residue S-Monoglycosylated GM2-Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library.

PubMed

Nakamura, Takahiro; Sato, Kohei; Naruse, Naoto; Kitakaze, Keisuke; Inokuma, Tsubasa; Hirokawa, Takatsugu; Shigenaga, Akira; Itoh, Kohji; Otaka, Akira

2016-10-17

A synthetic protocol for the preparation of 162-residue S-monoglycosylated GM2-activator protein (GM2AP) analogues bearing various amino acid substitutions for Thr69 has been developed. The facile incorporation of the replacements into the protein was achieved by means of a one-pot/N-to-C-directed sequential ligation strategy using readily accessible middle N-sulfanylethylanilide (SEAlide) peptides each consisting of seven amino acid residues. A kinetically controlled ligation protocol was successfully applied to the assembly of three peptide segments covering the GM2AP. The native chemical ligation (NCL) reactivities of the SEAlide peptides can be tuned by the presence or absence of phosphate salts. Furthermore, NCL of the alkyl thioester fragment [GM2AP (1-31)] with the N-terminal cysteinyl prolyl thioester [GM2AP (32-67)] proceeded smoothly to yield the 67-residue prolyl thioester, with the prolyl thioester moiety remaining intact. This newly developed strategy enabled the facile synthesis of GM2AP analogues. Thus, we refer to this synthetic protocol as "tailored synthesis" for the construction of a GM2AP library. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phage display screening without repetitious selection rounds.

PubMed

't Hoen, Peter A C; Jirka, Silvana M G; Ten Broeke, Bradley R; Schultes, Erik A; Aguilera, Begoña; Pang, Kar Him; Heemskerk, Hans; Aartsma-Rus, Annemieke; van Ommen, Gertjan J; den Dunnen, Johan T

2012-02-15

Phage display screenings are frequently employed to identify high-affinity peptides or antibodies. Although successful, phage display is a laborious technology and is notorious for identification of false positive hits. To accelerate and improve the selection process, we have employed Illumina next generation sequencing to deeply characterize the Ph.D.-7 M13 peptide phage display library before and after several rounds of biopanning on KS483 osteoblast cells. Sequencing of the naive library after one round of amplification in bacteria identifies propagation advantage as an important source of false positive hits. Most important, our data show that deep sequencing of the phage pool after a first round of biopanning is already sufficient to identify positive phages. Whereas traditional sequencing of a limited number of clones after one or two rounds of selection is uninformative, the required additional rounds of biopanning are associated with the risk of losing promising clones propagating slower than nonbinding phages. Confocal and live cell imaging confirms that our screen successfully selected a peptide with very high binding and uptake in osteoblasts. We conclude that next generation sequencing can significantly empower phage display screenings by accelerating the finding of specific binders and restraining the number of false positive hits. Copyright © 2011 Elsevier Inc. All rights reserved.

Guiding principles for peptide nanotechnology through directed discovery.

PubMed

Lampel, A; Ulijn, R V; Tuttle, T

2018-05-21

Life's diverse molecular functions are largely based on only a small number of highly conserved building blocks - the twenty canonical amino acids. These building blocks are chemically simple, but when they are organized in three-dimensional structures of tremendous complexity, new properties emerge. This review explores recent efforts in the directed discovery of functional nanoscale systems and materials based on these same amino acids, but that are not guided by copying or editing biological systems. The review summarises insights obtained using three complementary approaches of searching the sequence space to explore sequence-structure relationships for assembly, reactivity and complexation, namely: (i) strategic editing of short peptide sequences; (ii) computational approaches to predicting and comparing assembly behaviours; (iii) dynamic peptide libraries that explore the free energy landscape. These approaches give rise to guiding principles on controlling order/disorder, complexation and reactivity by peptide sequence design.

Powerful workhorses for antimicrobial peptide expression and characterization.

PubMed

Li, Chun; Blencke, Hans-Matti; Paulsen, Victoria; Haug, Tor; Stensvåg, Klara

2010-01-01

Discovery of antimicrobial peptides (AMP) is to a large extent based on screening of fractions of natural samples in bacterial growth inhibition assays. However, the use of bacteria is not limited to screening for antimicrobial substances. In later steps, bioengineered "bugs" can be applied to both production and characterization of AMPs. Here we describe the idea to use genetically modified Escherichia coli strains for both these purposes. This approach allowed us to investigate SpStrongylocins 1 and 2 from the purple sea urchin Strongylocentrotus purpuratus only based on sequence information from a cDNA library and without previous direct isolation or chemical synthesis of these peptides. The recombinant peptides are proved active against all bacterial strains tested. An assay based on a recombinant E. coli sensor strain expressing insect luciferase, revealed that SpStrongylocins are not interfering with membrane integrity and are therefore likely to have intracellular targets. © 2010 Landes Bioscience

Characterizing SH2 Domain Specificity and Network Interactions Using SPOT Peptide Arrays.

PubMed

Liu, Bernard A

2017-01-01

Src Homology 2 (SH2) domains are protein interaction modules that recognize and bind tyrosine phosphorylated ligands. Their ability to distinguish binding to over thousands of potential phosphotyrosine (pTyr) ligands within the cell is critical for the fidelity of receptor tyrosine kinase (RTK) signaling. Within humans there are over a hundred SH2 domains with more than several thousand potential ligands across many cell types and cell states. Therefore, defining the specificity of individual SH2 domains is critical for predicting and identifying their physiological ligands. Here, in this chapter, I describe the broad use of SPOT peptide arrays for examining SH2 domain specificity. An orientated peptide array library (OPAL) approach can uncover both favorable and non-favorable residues, thus providing an in-depth analysis to SH2 specificity. Moreover, I discuss the application of SPOT arrays for paneling SH2 ligand binding with physiological peptides.

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage

PubMed Central

Zygiel, Emily M.; Noren, Karen A.; Adamkiewicz, Marta A.; Aprile, Richard J.; Bowditch, Heather K.; Carroll, Christine L.; Cerezo, Maria Abigail S.; Dagher, Adelle M.; Hebert, Courtney R.; Hebert, Lauren E.; Mahame, Gloria M.; Milne, Stephanie C.; Silvestri, Kelly M.; Sutherland, Sara E.; Sylvia, Alexandria M.; Taveira, Caitlyn N.; VanValkenburgh, David J.; Noren, Christopher J.

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5’-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5’-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries. PMID:28445507

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage.

PubMed

Zygiel, Emily M; Noren, Karen A; Adamkiewicz, Marta A; Aprile, Richard J; Bowditch, Heather K; Carroll, Christine L; Cerezo, Maria Abigail S; Dagher, Adelle M; Hebert, Courtney R; Hebert, Lauren E; Mahame, Gloria M; Milne, Stephanie C; Silvestri, Kelly M; Sutherland, Sara E; Sylvia, Alexandria M; Taveira, Caitlyn N; VanValkenburgh, David J; Noren, Christopher J; Hall, Marilena Fitzsimons

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5'-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5'-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries.

An open-source computational and data resource to analyze digital maps of immunopeptidomes

PubMed Central

Caron, Etienne; Espona, Lucia; Kowalewski, Daniel J; Schuster, Heiko; Ternette, Nicola; Alpízar, Adán; Schittenhelm, Ralf B; Ramarathinam, Sri H; Lindestam Arlehamn, Cecilia S; Chiek Koh, Ching; Gillet, Ludovic C; Rabsteyn, Armin; Navarro, Pedro; Kim, Sangtae; Lam, Henry; Sturm, Theo; Marcilla, Miguel; Sette, Alessandro; Campbell, David S; Deutsch, Eric W; Moritz, Robert L; Purcell, Anthony W; Rammensee, Hans-Georg; Stevanovic, Stefan; Aebersold, Ruedi

2015-01-01

We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies. DOI: http://dx.doi.org/10.7554/eLife.07661.001 PMID:26154972

Cloning, expression, and purification of a new antimicrobial peptide gene from Musca domestica larva.

PubMed

Pei, Zhihua; Sun, Xiaoning; Tang, Yan; Wang, Kai; Gao, Yunhang; Ma, Hongxia

2014-10-01

Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram- bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study. Copyright © 2014 Elsevier B.V. All rights reserved.

Phylloseptin-PBa—A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity

PubMed Central

Wan, Yuantai; Ma, Chengbang; Zhou, Mei; Xi, Xinping; Li, Lei; Wu, Di; Wang, Lei; Lin, Chen; Lopez, Juan Chavez; Chen, Tianbao; Shaw, Chris

2015-01-01

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities. PMID:26633506

Phylloseptin-PBa--A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity.

PubMed

Wan, Yuantai; Ma, Chengbang; Zhou, Mei; Xi, Xinping; Li, Lei; Wu, Di; Wang, Lei; Lin, Chen; Lopez, Juan Chavez; Chen, Tianbao; Shaw, Chris

2015-12-01

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities.

Screening of Pro-Asp Sequences Exposed on Bacteriophage M13 as an Ideal Anchor for Gold Nanocubes.

PubMed

Lee, Hwa Kyoung; Lee, Yujean; Kim, Hyori; Lee, Hye-Eun; Chang, Hyejin; Nam, Ki Tae; Jeong, Dae Hong; Chung, Junho

2017-09-15

Bacteriophages are thought to be ideal vehicles for linking antibodies to nanoparticles. Here, we define the sequence of peptides exposed as a fusion protein on M13 bacteriophages to yield optimal binding of gold nanocubes and efficient bacteriophage amplification. We generated five helper bacteriophage libraries using AE(X) 2 DP, AE(X) 3 DP, AE(X) 4 DP, AE(X) 5 DP, and AE(X) 6 DP as the exposed portion of pVIII, in which X was a randomized amino acid residue encoded by the nucleotide sequence NNK. Efficient phage amplification was achievable only in the AE(X) 2 DP, AE(X) 3 DP, and AE(X) 4 DP libraries. Through biopanning with gold nanocubes, we enriched the phage clones and selected the clone with the highest fold change after enrichment. This clone displayed Pro-Asp on the surface of the bacteriophage and had amplification yields similar to those of the wild-type helper bacteriophage (VCSM13). The clone displayed even binding of gold nanocubes along its length and minimal aggregation after binding. We conclude that, for efficient amplification, the exposed pVIII amino acid length should be limited to six residues and Ala-Glu-Pro-Asp-Asp-Pro (AEPDDP) is the ideal fusion protein sequence for guaranteeing the optimal formation of a complex with gold nanocubes.

Structure-activity relationship of HP (2-20) analog peptide: enhanced antimicrobial activity by N-terminal random coil region deletion.

PubMed

Park, Yoonkyung; Park, Seong-Cheol; Park, Hae-Kyun; Shin, Song Yub; Kim, Yangmee; Hahm, Kyung-Soo

2007-01-01

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is a 19-aa antimicrobial peptide derived from N-terminus of Helicobacter pylori Ribosomal protein L1 (RpL1). In the previous study, several analogs with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, substitutions of Gln(16) and Asp(18) with Trp (Anal 3) for hydrophobic amino acid caused a dramatic increase in antibiotic activity without a hemolytic effect. HP-A3 is a potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 2-5) and extended C-terminal regular alpha-helical region (residues 6-20). To obtain the short and potent alpha-helical antimicrobial peptide, we synthesized a N-terminal random coil deleted HP-A3 (A3-NT) and examined their antimicrobial activity and mechanism of action. The resulting 15mer peptide showed increased antibacterial and antifungal activity to 2- and 4-fold, respectively, without hemolysis. Confocal fluorescence microscopy studies showed that A3-NT was accumulated in the plasma membrane. Flow cytometric analysis revealed that A3-NT acted in salt- and energy-independent manner. Furthermore, A3-NT causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy. Circular dichroism (CD) analysis revealed that A3-NT showed higher alpha-helical contents than the HP-A3 peptide in 50% TFE solution. Therefore, the cell-lytic efficiency of HP-A3, which depended on the alpha-helical content of peptide, correlated linearly with their antimicrobial potency.

Treatment with grass allergen peptides improves symptoms of grass pollen-induced allergic rhinoconjunctivitis.

PubMed

Ellis, Anne K; Frankish, Charles W; O'Hehir, Robyn E; Armstrong, Kristen; Steacy, Lisa; Larché, Mark; Hafner, Roderick P

2017-08-01

Synthetic peptide immunoregulatory epitopes are a new class of immunotherapy to treat allergic rhinoconjunctivitis (ARC). Grass allergen peptides, comprising 7 synthetic T-cell epitopes derived from Cyn d 1, Lol p 5, Dac g 5, Hol l 5, and Phl p 5, is investigated for treatment of grass pollen-induced ARC. We sought to evaluate the efficacy, safety, and tolerability of intradermally administered grass allergen peptides. A multicenter, randomized, double-blind, placebo-controlled study evaluated 3 regimens of grass allergen peptides versus placebo in patients with grass pollen-induced allergy (18-65 years). After a 4-day baseline challenge to rye grass in the environmental exposure unit (EEU), subjects were randomized to receive grass allergen peptides at 6 nmol at 2-week intervals for a total of 8 doses (8x6Q2W), grass allergen peptides at 12 nmol at 4-week intervals for a total of 4 doses (4x12Q4W), or grass allergen peptides at 12 nmol at 2-week intervals for a total of 8 doses (8x12Q2W) or placebo and treated before the grass pollen season. The primary efficacy end point was change from baseline in total rhinoconjunctivitis symptom score across days 2 to 4 of a 4-day posttreatment challenge (PTC) in the EEU after the grass pollen season. Secondary efficacy end points and safety were also assessed. Two hundred eighty-two subjects were randomized. Significantly greater improvement (reduction of total rhinoconjunctivitis symptom score from baseline to PTC) occurred across days 2 to 4 with grass allergen peptide 8x6Q2W versus placebo (-5.4 vs -3.8, respectively; P = .0346). Greater improvement at PTC also occurred for grass allergen peptide 8x6Q2W versus placebo (P = .0403) in patients with more symptomatic ARC. No safety signals were detected. Grass allergen peptide 8x6Q2W significantly improved ARC symptoms after rye grass allergen challenge in an EEU with an acceptable safety profile. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

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.

An In Vitro Translation, Selection, and Amplification System for Peptide Nucleic Acids

PubMed Central

Brudno, Yevgeny; Birnbaum, Michael E.; Kleiner, Ralph E.; Liu, David R.

2009-01-01

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include: (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template; (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures; (iii) selection of synthetic polymer libraries for desired binding or catalytic properties; and (iv) amplification of template sequences surviving selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of twelve PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection, and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory. PMID:20081830

A random forest learning assisted "divide and conquer" approach for peptide conformation search.

PubMed

Chen, Xin; Yang, Bing; Lin, Zijing

2018-06-11

Computational determination of peptide conformations is challenging as it is a problem of finding minima in a high-dimensional space. The "divide and conquer" approach is promising for reliably reducing the search space size. A random forest learning model is proposed here to expand the scope of applicability of the "divide and conquer" approach. A random forest classification algorithm is used to characterize the distributions of the backbone φ-ψ units ("words"). A random forest supervised learning model is developed to analyze the combinations of the φ-ψ units ("grammar"). It is found that amino acid residues may be grouped as equivalent "words", while the φ-ψ combinations in low-energy peptide conformations follow a distinct "grammar". The finding of equivalent words empowers the "divide and conquer" method with the flexibility of fragment substitution. The learnt grammar is used to improve the efficiency of the "divide and conquer" method by removing unfavorable φ-ψ combinations without the need of dedicated human effort. The machine learning assisted search method is illustrated by efficiently searching the conformations of GGG/AAA/GGGG/AAAA/GGGGG through assembling the structures of GFG/GFGG. Moreover, the computational cost of the new method is shown to increase rather slowly with the peptide length.

Novel ligands for cancer diagnosis: selection of peptide ligands for identification and isolation of B-cell lymphomas.

PubMed

McGuire, Michael J; Samli, Kausar N; Chang, Ya-Ching; Brown, Kathlynn C

2006-04-01

Lymphoma and leukemia account for nearly 8% of cancer fatalities each year. Present treatments do not differentiate between normal and malignant cells. New reagents that distinguish malignant cells and enable the isolation of these cells from the normal background will enhance the molecular characterization of disease and specificity of treatment. Peptide ligands were selected from a phage-displayed peptide library by biopanning on the B-cell lymphoma line, A20. The isolated peptides were assessed as reagents for identification and isolation of lymphoma cells by flow cytometry and cell capture with magnetic beads. Two novel peptides and one obtained previously on cardiomyocytes were selected. A20 cells bind phage displaying these peptides 250- to 450-fold over control phage. These phage bind to other bone marrow-derived cancel lines including some macrophage and T cells but do not bind to normal splenocytes. Synthetic constructs of these peptides have binding affinities comparable to B-cell-specific antibodies. Similar to antibodies, these peptides can be used in flow cytometry and magnetic bead capture to distinguish lymphoma cells from normal splenocytes. Bone marrow-derived malignant cells express cell surface markers that can be used to distinguish them from normal cells. These results demonstrate the ability to use an unbiased screen to rapidly generate high-affinity peptide ligands for identification and isolation of lymphoma cells.

Functionalization of semiconductors for biosensing applications

NASA Astrophysics Data System (ADS)

Estephan, E.; Larroque, C.; Martineau, P.; Cloitre, T.; Gergely, Cs.

2007-05-01

Functionalization of semiconductors (SC) has been widely used for various electronic, photonic and biomedical applications. In this paper, we report on selective functionalization achieved by peptides that reveal specific recognition of the SC surfaces. A M13 bacteriophage library was used to screen 10 10 different 12-mer peptide on various SC substrates to successfully isolate after 3 cycles one specific peptide for the majority of semiconductors. Our results conclude that GaAs(100) and GaN(0001) retain the same sequence of 12-mer peptide, suggesting that the specificity does not depend on the crystallographic structure but it depends on the chemical composition and the electronegativity of the surface, thus on the orientation of the material. We also note the presence of at least one proline (Pro) amino acid in each peptide, and the presence of the histidine (His) in the specific peptides for the II-VI class SC. Pro imprints a constraint to the peptide to facilitate adhesion to the surface, whereas the basic side chain His is known for its affinity towards some of the elements of class II SC. Finally, fluorescence microscopy has been employed to demonstrate the preferential attachment of the peptide to their specific SC surface in close proximity to a surface of different chemical and structural composition. The use of selected peptides expressed by phage display can be extended to encompass a variety of nanostructured semiconductor based devices.

Isolation and characterization of lymphocyte-like cells from a lamprey

PubMed Central

Mayer, Werner E.; Uinuk-ool, Tatiana; Tichy, Herbert; Gartland, Lanier A.; Klein, Jan; Cooper, Max D.

2002-01-01

Lymphocyte-like cells in the intestine of the sea lamprey, Petromyzon marinus, were isolated by flow cytometry under light-scatter conditions used for the purification of mouse intestinal lymphocytes. The purified lamprey cells were morphologically indistinguishable from mammalian lymphocytes. A cDNA library was prepared from the lamprey lymphocyte-like cells, and more than 8,000 randomly selected clones were sequenced. Homology searches comparing these ESTs with sequences deposited in the databases led to the identification of numerous genes homologous to those predominantly or characteristically expressed in mammalian lymphocytes, which included genes controlling lymphopoiesis, intracellular signaling, proliferation, migration, and involvement of lymphocytes in innate immune responses. Genes closely related to those that in gnathostomes control antigen processing and transport of antigenic peptides could be ascertained, although no sequences with significant similarity to MHC, T cell receptor, or Ig genes were found. The data suggest that the evolution of lymphocytes in the lamprey has reached a stage poised for the emergence of adaptive immunity. PMID:12388781

Meta-genome-wide association studies identify a locus on chromosome 1 and multiple variants in the MHC region for serum C-peptide in type 1 diabetes.

PubMed

Roshandel, Delnaz; Gubitosi-Klug, Rose; Bull, Shelley B; Canty, Angelo J; Pezzolesi, Marcus G; King, George L; Keenan, Hillary A; Snell-Bergeon, Janet K; Maahs, David M; Klein, Ronald; Klein, Barbara E K; Orchard, Trevor J; Costacou, Tina; Weedon, Michael N; Oram, Richard A; Paterson, Andrew D

2018-05-01

The aim of this study was to identify genetic variants associated with beta cell function in type 1 diabetes, as measured by serum C-peptide levels, through meta-genome-wide association studies (meta-GWAS). We performed a meta-GWAS to combine the results from five studies in type 1 diabetes with cross-sectionally measured stimulated, fasting or random C-peptide levels, including 3479 European participants. The p values across studies were combined, taking into account sample size and direction of effect. We also performed separate meta-GWAS for stimulated (n = 1303), fasting (n = 2019) and random (n = 1497) C-peptide levels. In the meta-GWAS for stimulated/fasting/random C-peptide levels, a SNP on chromosome 1, rs559047 (Chr1:238753916, T>A, minor allele frequency [MAF] 0.24-0.26), was associated with C-peptide (p = 4.13 × 10 -8 ), meeting the genome-wide significance threshold (p < 5 × 10 -8 ). In the same meta-GWAS, a locus in the MHC region (rs9260151) was close to the genome-wide significance threshold (Chr6:29911030, C>T, MAF 0.07-0.10, p = 8.43 × 10 -8 ). In the stimulated C-peptide meta-GWAS, rs61211515 (Chr6:30100975, T/-, MAF 0.17-0.19) in the MHC region was associated with stimulated C-peptide (β [SE] = - 0.39 [0.07], p = 9.72 × 10 -8 ). rs61211515 was also associated with the rate of stimulated C-peptide decline over time in a subset of individuals (n = 258) with annual repeated measures for up to 6 years (p = 0.02). In the meta-GWAS of random C-peptide, another MHC region, SNP rs3135002 (Chr6:32668439, C>A, MAF 0.02-0.06), was associated with C-peptide (p = 3.49 × 10 -8 ). Conditional analyses suggested that the three identified variants in the MHC region were independent of each other. rs9260151 and rs3135002 have been associated with type 1 diabetes, whereas rs559047 and rs61211515 have not been associated with a risk of developing type 1 diabetes. We identified a locus on chromosome 1 and multiple variants in the MHC region, at least some of which were distinct from type 1 diabetes risk loci, that were associated with C-peptide, suggesting partly non-overlapping mechanisms for the development and progression of type 1 diabetes. These associations need to be validated in independent populations. Further investigations could provide insights into mechanisms of beta cell loss and opportunities to preserve beta cell function.

McDonald and Company Securities Library User Survey, 1996.

ERIC Educational Resources Information Center

Wolfgram, Derek E.

The library of McDonald and Company Securities is important to the success of the business and its employees. This study assesses the needs and expectations of the library users, and analyzes how well the current library services are meeting those needs and expectations. A questionnaire was distributed to a large random sample of the firm's…

Effects of Transdermal Testosterone on Natriuretic Peptide Levels in Women: A Randomized Placebo-Controlled Pilot Study

PubMed Central

Lin, Eleanor; McCabe, Elizabeth; Newton-Cheh, Christopher; Bloch, Kenneth; Buys, Emmanuel; Wang, Thomas; Miller, Karen K.

2011-01-01

Objective To investigate whether testosterone administration alters natriuretic peptide levels in women. Design Three-month, double-blind, randomized, placebo-controlled study. Setting Clinical research center. Patients 51 women with hypoandrogenemia due to hypopituitarism. Intervention Transdermal testosterone (300 mcg daily) or placebo patch. Main Outcome Measure N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels. Results NT-proBNP levels decreased in the transdermal testosterone group compared with placebo over three months (p = 0.009). The difference between groups remained significant after controlling for baseline age, systolic blood pressure, body mass index, and homeostasis model of assessment-insulin resistance (p = 0.008). Change in NT-proBNP over three months was inversely associated with change in free testosterone levels (ρ = −0.41, p = 0.01). Conclusions Testosterone administration to women results in decreased natriuretic peptide levels, suggesting that testosterone may be an inverse regulator of the natriuretic peptide system. Clinical Trials Registration Number NCT00027430 PMID:22137497

HomoSAR: bridging comparative protein modeling with quantitative structural activity relationship to design new peptides.

PubMed

Borkar, Mahesh R; Pissurlenkar, Raghuvir R S; Coutinho, Evans C

2013-11-15

Peptides play significant roles in the biological world. To optimize activity for a specific therapeutic target, peptide library synthesis is inevitable; which is a time consuming and expensive. Computational approaches provide a promising way to simply elucidate the structural basis in the design of new peptides. Earlier, we proposed a novel methodology termed HomoSAR to gain insight into the structure activity relationships underlying peptides. Based on an integrated approach, HomoSAR uses the principles of homology modeling in conjunction with the quantitative structural activity relationship formalism to predict and design new peptide sequences with the optimum activity. In the present study, we establish that the HomoSAR methodology can be universally applied to all classes of peptides irrespective of sequence length by studying HomoSAR on three peptide datasets viz., angiotensin-converting enzyme inhibitory peptides, CAMEL-s antibiotic peptides, and hAmphiphysin-1 SH3 domain binding peptides, using a set of descriptors related to the hydrophobic, steric, and electronic properties of the 20 natural amino acids. Models generated for all three datasets have statistically significant correlation coefficients (r(2)) and predictive r2 (r(pred)2) and cross validated coefficient ( q(LOO)2). The daintiness of this technique lies in its simplicity and ability to extract all the information contained in the peptides to elucidate the underlying structure activity relationships. The difficulties of correlating both sequence diversity and variation in length of the peptides with their biological activity can be addressed. The study has been able to identify the preferred or detrimental nature of amino acids at specific positions in the peptide sequences. Copyright © 2013 Wiley Periodicals, Inc.

Targeting the Atypical Chemokine Receptor ACKR3/CXCR7: Phase 1 - Phage Display Peptide Identification and Characterization.

PubMed

Vestal, R D; LaJeunesse, D R; Taylor, E W

2016-01-01

One of the greatest challenges in fighting cancer is cell targeting and biomarker selection. The Atypical Chemokine Receptor ACKR3/CXCR7 is expressed on many cancer cell types, including breast cancer and glioblastoma, and binds the endogenous ligands SDF1/CXCL12 and ITAC/CXCL11. A 20 amino acid region of the ACKR3/CXCR7 N-terminus was synthesized and targeted with the NEB PhD-7 Phage Display Peptide Library. Twenty-nine phages were isolated and heptapeptide inserts sequenced; of these, 23 sequences were unique. A 3D molecular model was created for the ACKR3/CXCR7 N-terminus by mutating the corresponding region of the crystal structure of CXCR4 with bound SDF1/CXCL12. A ClustalW alignment was performed on each peptide sequence using the entire SDF1/CXCL12 sequence as the template. The 23-peptide sequences showed similarity to three distinct regions of the SDF1/CXCL12 molecule. A 3D molecular model was made for each of the phage peptide inserts to visually identify potential areas of steric interference of peptides that simulated CXCL12 regions not in contact with the receptor's Nterminus. An ELISA analysis of the relative binding affinity between the peptides identified 9 peptides with statistically significant results. The candidate pool of 9 peptides was further reduced to 3 peptides based on their affinity for the targeted N-terminus region peptide versus no target peptide present or a scrambled negative control peptide. The results clearly show the Phage Display protocol can be used to target a synthesized region of the ACKR3/CXCR7 N-terminus. The 3 peptides chosen, P20, P3, and P9, will be the basis for further targeting studies.

Combinatorial Approach for Large-scale Identification of Linked Peptides from Tandem Mass Spectrometry Spectra*

PubMed Central

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

2014-01-01

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

Benefits and Harms of Sick Leave: Lack of Randomized, Controlled Trials

ERIC Educational Resources Information Center

Axelsson, Inge; Marnetoft, Sven-Uno

2010-01-01

The aim of this study was to try to identify those randomized controlled trials that compare sick leave with no sick leave or a different duration or degree of sick leave. A comprehensive, systematic, electronic search of Clinical Evidence, the Cochrane Library and PubMed, and a manual search of the Campbell Library and a journal supplement was…

SABinder: A Web Service for Predicting Streptavidin-Binding Peptides.

PubMed

He, Bifang; Kang, Juanjuan; Ru, Beibei; Ding, Hui; Zhou, Peng; Huang, Jian

2016-01-01

Streptavidin is sometimes used as the intended target to screen phage-displayed combinatorial peptide libraries for streptavidin-binding peptides (SBPs). More often in the biopanning system, however, streptavidin is just a commonly used anchoring molecule that can efficiently capture the biotinylated target. In this case, SBPs creeping into the biopanning results are not desired binders but target-unrelated peptides (TUP). Taking them as intended binders may mislead subsequent studies. Therefore, it is important to find if a peptide is likely to be an SBP when streptavidin is either the intended target or just the anchoring molecule. In this paper, we describe an SVM-based ensemble predictor called SABinder. It is the first predictor for SBP. The model was built with the feature of optimized dipeptide composition. It was observed that 89.20% (MCC = 0.78; AUC = 0.93; permutation test, p < 0.001) of peptides were correctly classified. As a web server, SABinder is freely accessible. The tool provides a highly efficient way to exclude potential SBP when they are TUP or to facilitate identification of possibly new SBP when they are the desired binders. In either case, it will be helpful and can benefit related scientific community.

Monitoring multiple myeloma by idiotype-specific peptide binders of tumor-derived exosomes.

PubMed

Iaccino, Enrico; Mimmi, Selena; Dattilo, Vincenzo; Marino, Fabiola; Candeloro, Patrizio; Di Loria, Antonio; Marimpietri, Danilo; Pisano, Antonio; Albano, Francesco; Vecchio, Eleonora; Ceglia, Simona; Golino, Gaetanina; Lupia, Antonio; Fiume, Giuseppe; Quinto, Ileana; Scala, Giuseppe

2017-10-13

Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression.

Comparative Analyses of Data Independent Acquisition Mass Spectrometric Approaches: DIA, WiSIM‐DIA, and Untargeted DIA

PubMed Central

Ho, Jenny T. C.; Smit, August B.; Li, Ka Wan

2018-01-01

Abstract Data‐independent acquisition (DIA) is an emerging technology for quantitative proteomics. Current DIA focusses on the identification and quantitation of fragment ions that are generated from multiple peptides contained in the same selection window of several to tens of m/z. An alternative approach is WiSIM‐DIA, which combines conventional DIA with wide‐SIM (wide selected‐ion monitoring) windows to partition the precursor m/z space to produce high‐quality precursor ion chromatograms. However, WiSIM‐DIA has been underexplored; it remains unclear if it is a viable alternative to DIA. We demonstrate that WiSIM‐DIA quantified more than 24 000 unique peptides over five orders of magnitude in a single 2 h analysis of a neuronal synapse‐enriched fraction, compared to 31 000 in DIA. There is a strong correlation between abundance values of peptides quantified in both the DIA and WiSIM‐DIA datasets. Interestingly, the S/N ratio of these peptides is not correlated. We further show that peptide identification directly from DIA spectra identified >2000 proteins, which included unique peptides not found in spectral libraries generated by DDA. PMID:29134766

Aminopeptidase A is a functional target in angiogenic blood vessels.

PubMed

Marchiò, Serena; Lahdenranta, Johanna; Schlingemann, Reinier O; Valdembri, Donatella; Wesseling, Pieter; Arap, Marco A; Hajitou, Amin; Ozawa, Michael G; Trepel, Martin; Giordano, Ricardo J; Nanus, David M; Dijkman, Henri B P M; Oosterwijk, Egbert; Sidman, Richard L; Cooper, Max D; Bussolino, Federico; Pasqualini, Renata; Arap, Wadih

2004-02-01

We show that a membrane-associated protease, aminopeptidase A (APA), is upregulated and enzymatically active in blood vessels of human tumors. To gain mechanistic insight, we evaluated angiogenesis in APA null mice. We found that, although these mice develop normally, they fail to mount the expected angiogenic response to hypoxia or growth factors. We then isolated peptide inhibitors of APA from a peptide library and show that they specifically bind to and inhibit APA, suppress migration and proliferation of endothelial cells, inhibit angiogenesis, and home to tumor blood vessels. Finally, we successfully treated tumor-bearing mice with APA binding peptides or anti-APA blocking monoclonal antibodies. These data show that APA is a regulator of blood vessel formation, and can serve as a functional vascular target.

Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels.

PubMed

Deshmukh, Ameya A; Weist, Jessica L; Leight, Jennifer L

2018-05-01

Current zymographic techniques detect only a subset of known proteases due to the limited number of native proteins that have been optimized for incorporation into polyacrylamide gels. To address this limitation, we have developed a technique to covalently incorporate fluorescently labeled, protease-sensitive peptides using an azido-PEG3-maleimide crosslinker. Peptides incorporated into gels enabled measurement of MMP-2, -9, -14, and bacterial collagenase. Sensitivity analysis demonstrated that use of peptide functionalized gels could surpass detection limits of current techniques. Finally, electrophoresis of conditioned media from cultured cells resulted in the appearance of several proteolytic bands, some of which were undetectable by gelatin zymography. Taken together, these results demonstrate that covalent incorporation of fluorescent substrates can greatly expand the library of detectable proteases using zymographic techniques.

On the Split Personality of Penultimate Proline

PubMed Central

Glover, Matthew S.; Shi, Liuqing; Fuller, Daniel R.; Arnold, Randy J.; Radivojac, Predrag; Clemmer, David E.

2014-01-01

The influence of the position of the amino acid proline in polypeptide sequences is examined by a combination of ion mobility spectrometry-mass spectrometry (IMS-MS), amino acid substitutions, and molecular modeling. The results suggest that when proline exists as the second residue from the N-terminus (i.e., penultimate proline), two families of conformers are formed. We demonstrate the existence of these families by a study of a series of truncated and mutated peptides derived from the 11-residue peptide Ser1-Pro2-Glu3-Leu4-Pro5-Ser6-Pro7-Gln8-Ala9-Glu10-Lys11. We find that every peptide from this sequence with a penultimate proline residue has multiple conformations. Substitution of Ala for Pro residues indicates that multiple conformers arise from the cis- trans isomerization of Xaa1–Pro2 peptide bonds as Xaa–Ala peptide bonds are unlikely to adopt the cis isomer, and examination of spectra from a library of 58 peptides indicates that ~80% of sequences show this effect. A simple mechanism suggesting that the barrier between the cis-and trans-proline forms is lowered because of low steric impedance is proposed. This observation may have interesting biological implications as well, and we note that a number of biologically active peptides have penultimate proline residues. PMID:25503299

The Degree and Nature to Which Public School Libraries Are Automated: A Survey of Public School Libraries in Ohio.

ERIC Educational Resources Information Center

Meckler, Elizabeth M.

This paper examines the belief that no more than half of the public school libraries in the state of Ohio are automated to any degree. The purpose of the research was to determine the degree and nature of automation at the public school libraries in Ohio. A written survey was mailed to 350 libraries that represented a randomized sample of the…

Serum Stabilities of Short Tryptophan- and Arginine-Rich Antimicrobial Peptide Analogs

PubMed Central

Nguyen, Leonard T.; Chau, Johnny K.; Perry, Nicole A.; de Boer, Leonie; Zaat, Sebastian A. J.; Vogel, Hans J.

2010-01-01

Background Several short antimicrobial peptides that are rich in tryptophan and arginine residues were designed with a series of simple modifications such as end capping and cyclization. The two sets of hexapeptides are based on the Trp- and Arg-rich primary sequences from the “antimicrobial centre” of bovine lactoferricin as well as an antimicrobial sequence obtained through the screening of a hexapeptide combinatorial library. Methodology/Principal Findings HPLC, mass spectrometry and antimicrobial assays were carried out to explore the consequences of the modifications on the serum stability and microbicidal activity of the peptides. The results show that C-terminal amidation increases the antimicrobial activity but that it makes little difference to its proteolytic degradation in human serum. On the other hand, N-terminal acetylation decreases the peptide activities but significantly increases their protease resistance. Peptide cyclization of the hexameric peptides was found to be highly effective for both serum stability and antimicrobial activity. However the two cyclization strategies employed have different effects, with disulfide cyclization resulting in more active peptides while backbone cyclization results in more proteolytically stable peptides. However, the benefit of backbone cyclization did not extend to longer 11-mer peptides derived from the same region of lactoferricin. Mass spectrometry data support the serum stability assay results and allowed us to determine preferred proteolysis sites in the peptides. Furthermore, isothermal titration calorimetry experiments showed that the peptides all had weak interactions with albumin, the most abundant protein in human serum. Conclusions/Significance Taken together, the results provide insight into the behavior of the peptides in human serum and will therefore aid in advancing antimicrobial peptide design towards systemic applications. PMID:20844765

In-silico screening and validation of high-affinity tetra-peptide inhibitor of Leishmania donovani O-acetyl serine sulfhydrylase (OASS).

PubMed

Kant, Vishnu; Vijayakumar, Saravanan; Sahoo, Ganesh Chandra; Chaudhery, Shailendra S; Das, Pradeep

2018-02-07

OASS is a specific enzyme that helps Leishmania parasite to survive the oxidative stress condition in human macrophages. SAT C-terminal peptides in several organisms, including Leishmania, were reported to inhibit or reduce the activity of OASS. Small peptide and small molecules mimicking the SAT C-terminal residues are designed and tested for the inhibition of OASS in different organisms. Hence, in this study, all the possible tetra-peptide combinations were designed and screened based on the docking ability with Leishmania donovani OASS (Ld-OASS). The top ranked peptides were further validated for the stability using 50 ns molecular dynamic simulation. In order to identify the better binding capability of the peptides, the top peptides complexed with Ld-OASS were also subjected to molecular dynamic simulation. The docking and simulation results favored the peptide EWSI to possess greater advantage than previously reported peptide (DWSI) in binding with Ld-OASS active site. Also, screening of non-peptide inhibitor of Asinex Biodesign library based on the shape similarity of EWSI and DWSI was performed. The top similar molecules of each peptides were docked on to Ld-OASS active site and subsequently simulated for 20 ns. The results suggested that the ligand that shares high shape similarity with EWSI possess better binding capability than the ligand that shares high shape similarity with DWSI. This study revealed that the tetra-peptide EWSI had marginal advantage over DWSI in binding with Ld-OASS, thereby providing basis for defining a pharmacophoric scaffold for the design of peptidomimetic inhibitors as well as non-peptide inhibitors of Ld-OASS.

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

PubMed Central

David, Marion; Lécorché, Pascaline; Masse, Maxime; Faucon, Aude; Abouzid, Karima; Gaudin, Nicolas; Varini, Karine; Gassiot, Fanny; Ferracci, Géraldine; Jacquot, Guillaume; Vlieghe, Patrick

2018-01-01

Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells—or organs that express the LDLR. PMID:29485998

Proteasix: a tool for automated and large-scale prediction of proteases involved in naturally occurring peptide generation.

PubMed

Klein, Julie; Eales, James; Zürbig, Petra; Vlahou, Antonia; Mischak, Harald; Stevens, Robert

2013-04-01

In this study, we have developed Proteasix, an open-source peptide-centric tool that can be used to predict in silico the proteases involved in naturally occurring peptide generation. We developed a curated cleavage site (CS) database, containing 3500 entries about human protease/CS combinations. On top of this database, we built a tool, Proteasix, which allows CS retrieval and protease associations from a list of peptides. To establish the proof of concept of the approach, we used a list of 1388 peptides identified from human urine samples, and compared the prediction to the analysis of 1003 randomly generated amino acid sequences. Metalloprotease activity was predominantly involved in urinary peptide generation, and more particularly to peptides associated with extracellular matrix remodelling, compared to proteins from other origins. In comparison, random sequences returned almost no results, highlighting the specificity of the prediction. This study provides a tool that can facilitate linking of identified protein fragments to predicted protease activity, and therefore into presumed mechanisms of disease. Experiments are needed to confirm the in silico hypotheses; nevertheless, this approach may be of great help to better understand molecular mechanisms of disease, and define new biomarkers, and therapeutic targets. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Hao; Dranchak, Patricia; Li, Zhiru

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanismmore » placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.« less

Molecular cloning of a cDNA encoding the precursor of adenoregulin from frog skin. Relationships with the vertebrate defensive peptides, dermaseptins.

PubMed

Amiche, M; Ducancel, F; Lajeunesse, E; Boulain, J C; Ménez, A; Nicolas, P

1993-03-31

Adenoregulin has recently been isolated from Phyllomedusa skin as a 33 amino acid residues peptide which enhanced binding of agonists to the A1 adenosine receptor. In order to study the structure of the precursor of adenoregulin we constructed a cDNA library from mRNAs extracted from the skin of Phyllomedusa bicolor. We detected the complete nucleotide sequence of a cDNA encoding the adenoregulin biosynthetic precursor. The deduced sequence of the precursor is 81 amino acids long, exhibits a putative signal sequence at the NH2 terminus and contains a single copy of the biologically active peptide at the COOH terminus. Structural and conformational homologies that are observed between adenoregulin and the dermaseptins, antimicrobial peptides exhibiting strong membranolytic activities against various pathogenic agents, suggest that adenoregulin is an additional member of the growing family of cytotropic antimicrobial peptides that allow vertebrate animals to defend themselves against microorganisms. As such, the adenosine receptor regulating activity of adenoregulin could be due to its ability to interact with and disrupt membranes lipid bilayers.

Characterization of the branched antimicrobial peptide M6 by analyzing its mechanism of action and in vivo toxicity.

PubMed

Pini, Alessandro; Giuliani, Andrea; Falciani, Chiara; Fabbrini, Monica; Pileri, Silvia; Lelli, Barbara; Bracci, Luisa

2007-06-01

We analyzed functional activity of the antimicrobial peptide M6 in vitro and in vivo. The peptide was identified by our group by phage library selection, rational modification and synthesis in a tetrabranched form (Pini et al., Antimicrob. Agents Chemother. 2005; 49: 2665-72). We found that it binds lipopolysaccharide, causes perforation of cell membranes without destroying external cell morphology and strongly binds DNA. The latter feature suggests that it could inhibit metabolic pathways, blocking DNA replication and/or transcription. We also observed that M6 does not stimulate humoral immune response when repeatedly administered to animals. We also analyzed M6 toxicity when administered to animals by intraperitoneal or by intravenous injection, determining a preliminary LD50 (125 and 37.5 mg/kg, respectively), which suggested that M6 could be used in vivo. These features make the antimicrobial branched peptide M6 a promising candidate for the development of a new antibacterial drug. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Designing Antibacterial Peptides with Enhanced Killing Kinetics

PubMed Central

Waghu, Faiza H.; Joseph, Shaini; Ghawali, Sanket; Martis, Elvis A.; Madan, Taruna; Venkatesh, Kareenhalli V.; Idicula-Thomas, Susan

2018-01-01

Antimicrobial peptides (AMPs) are gaining attention as substitutes for antibiotics in order to combat the risk posed by multi-drug resistant pathogens. Several research groups are engaged in design of potent anti-infective agents using natural AMPs as templates. In this study, a library of peptides with high sequence similarity to Myeloid Antimicrobial Peptide (MAP) family were screened using popular online prediction algorithms. These peptide variants were designed in a manner to retain the conserved residues within the MAP family. The prediction algorithms were found to effectively classify peptides based on their antimicrobial nature. In order to improve the activity of the identified peptides, molecular dynamics (MD) simulations, using bilayer and micellar systems could be used to design and predict effect of residue substitution on membranes of microbial and mammalian cells. The inference from MD simulation studies well corroborated with the wet-lab observations indicating that MD-guided rational design could lead to discovery of potent AMPs. The effect of the residue substitution on membrane activity was studied in greater detail using killing kinetic analysis. Killing kinetics studies on Gram-positive, negative and human erythrocytes indicated that a single residue change has a drastic effect on the potency of AMPs. An interesting outcome was a switch from monophasic to biphasic death rate constant of Staphylococcus aureus due to a single residue mutation in the peptide. PMID:29527201

Fission yield covariances for JEFF: A Bayesian Monte Carlo method

NASA Astrophysics Data System (ADS)

Leray, Olivier; Rochman, Dimitri; Fleming, Michael; Sublet, Jean-Christophe; Koning, Arjan; Vasiliev, Alexander; Ferroukhi, Hakim

2017-09-01

The JEFF library does not contain fission yield covariances, but simply best estimates and uncertainties. This situation is not unique as all libraries are facing this deficiency, firstly due to the lack of a defined format. An alternative approach is to provide a set of random fission yields, themselves reflecting covariance information. In this work, these random files are obtained combining the information from the JEFF library (fission yields and uncertainties) and the theoretical knowledge from the GEF code. Examples of this method are presented for the main actinides together with their impacts on simple burn-up and decay heat calculations.

Switchable Hydrolase Based on Reversible Formation of Supramolecular Catalytic Site Using a Self-Assembling Peptide.

PubMed

Zhang, Chunqiu; Shafi, Ramim; Lampel, Ayala; MacPherson, Douglas; Pappas, Charalampos G; Narang, Vishal; Wang, Tong; Maldarelli, Charles; Ulijn, Rein V

2017-11-13

The reversible regulation of catalytic activity is a feature found in natural enzymes which is not commonly observed in artificial catalytic systems. Here, we fabricate an artificial hydrolase with pH-switchable activity, achieved by introducing a catalytic histidine residue at the terminus of a pH-responsive peptide. The peptide exhibits a conformational transition from random coil to β-sheet by changing the pH from acidic to alkaline. The β-sheet self-assembles to form long fibrils with the hydrophobic edge and histidine residues extending in an ordered array as the catalytic microenvironment, which shows significant esterase activity. Catalytic activity can be reversible switched by pH-induced assembly/disassembly of the fibrils into random coils. At higher concentrations, the peptide forms a hydrogel which is also catalytically active and maintains its reversible (de-)activation. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discovery and Development of Synthetic and Natural Biomaterials for Protein Therapeutics and Medical Device Applications

NASA Astrophysics Data System (ADS)

Keefe, Andrew J.

Controlling nonspecific protein interactions is important for applications from medical devices to protein therapeutics. The presented work is a compilation of efforts aimed at using zwitterionic (ionic yet charge neutral) polymers to modify and stabilize the surface of sensitive biomedical and biological materials. Traditionally, when modifying the surface of a material, the stability of the underlying substrate. The materials modified in this dissertation are unique due to their unconventional amorphous characteristics which provide additional challenges. These are poly(dimethyl siloxane) (PDMS) rubber, and proteins. These materials may seem dissimilar, but both have amorphous surfaces, that do not respond well to chemical modification. PDMS is a biomaterial extensively used in medical device manufacturing, but experiences unacceptably high levels of non-specific protein fouling when used with biological samples. To reduce protein fouling, surface modification is often needed. Unfortunately conventional surface modification methods, such as Poly(ethylene glycol) (PEG) coatings, do not work for PDMS due to its amorphous state. Herein, we demonstrate how a superhydrophilic zwitterionic material, poly(carboxybetaine methacrylate) (pCBMA), can provide a highly stable nonfouling coating with long term stability due to the sharp the contrast in hydrophobicity between pCBMA and PDMS. Biological materials, such as proteins, also require stabilization to improve shelf life, circulation time, and bioactivity. Conjugation of proteins with PEG is often used to increase protein stability, but has a detrimental effect on bioactivity. Here we have shown that pCBMA conjugation improves stability in a similar fashion to PEG, but also retains, or even improves, binding affinity due to enhanced protein-substrate hydrophobic interactions. Recognizing that pCBMA chemically resembles the combination of lysine (K) and glutamic acid (E) amino acids, we have shown how zwitterionic nonfouling peptides can be genetically engineered onto a protein to form recombinant protein-polymer conjugates. This technique avoids the need to post-modify proteins, that is often expensive and time consuming in protein manufacturing. Finally, we have developed two new peptide screening methods that were able to select for nonfouling peptide sequences. The selection for nonfouling sequences is not possible using traditional methods (phage display, yeast display, bacterial display and resin display) due to the presence of background interference. In our first nonfouling peptide screening method, we measured the fouling properties of peptides that were immobilized on the surface of solid glass beads. Peptides first needed to be rationally designed, and then subsequently evaluated for protein binding. Using this method, we were able to screen of 10's of sequences. Our second nonfouling peptide screening method is able to screen thousands of peptide sequences using a combinatorially generated peptide library. This was accomplished using controlled pore glass (CPG) beads as substrates to develop one-bead-one-compound (OBOC) peptide libraries. The choice of a porous substrate made it possible to synthesize enough peptide material to allow for peptide sequencing from a single bead using mass spectrometry techniques.

Effects of side group functionality and molecular weight on the activity of synthetic antimicrobial polypeptides.

PubMed

Engler, Amanda C; Shukla, Anita; Puranam, Sravanthi; Buss, Hilda G; Jreige, Nina; Hammond, Paula T

2011-05-09

The rapid emergence of antibiotic-resistant bacteria along with increasing difficulty in biofilm treatment has caused an immediate need for the development of new classes of antimicrobial therapeutics. We have developed a library of antimicrobial polypeptides, prepared by the ring-opening polymerization of γ-propargyl-L-glutamate N-carboxyanhydride and the alkyne-azide cycloaddition click reaction, which mimic the favorable characteristics of naturally occurring antimicrobial peptides (AmPs). AmPs are known not to cause drug resistance as well as prevent bacteria attachment on surfaces. The ease and scale of synthesis of the antimicrobial polypeptides developed here are significantly improved over the traditional Merrifield synthetic peptide approaches needed for naturally occurring antimicrobial peptides and avoids the unique challenges of biosynthetic pathways. The polypeptides range in length from 30 to 140 repeat units and can have varied side group functionality, including primary, secondary, tertiary, and quaternary amines with hydrocarbon side chains ranging from 1 to 12 carbons long. Overall, we find these polypeptides to exhibit broad-spectrum activity against both Gram positive and Gram negative bacteria, namely, S. aureus and E. coli , while having very low hemolytic activity. Many of the polypeptides can also be used as surface coatings to prevent bacterial attachment. The polypeptide library developed in this work addresses the need for effective biocompatible therapeutics for drug delivery and medical device coatings.

Substrate specificity of mitochondrial intermediate peptidase analysed by a support-bound peptide library

PubMed Central

Marcondes, M.F.M.; Alves, F.M.; Assis, D.M.; Hirata, I.Y.; Juliano, L.; Oliveira, V.; Juliano, M.A.

2015-01-01

The substrate specificity of recombinant human mitochondrial intermediate peptidase (hMIP) using a synthetic support-bound FRET peptide library is presented. The collected fluorescent beads, which contained the hydrolysed peptides generated by hMIP, were sequenced by Edman degradation. The results showed that this peptidase presents a remarkable preference for polar uncharged residues at P1 and P1′ substrate positions: Ser = Gln > Thr at P1 and Ser > Thr at P1′. Non-polar residues were frequent at the substrate P3, P2, P2′ and P3′ positions. Analysis of the predicted MIP processing sites in imported mitochondrial matrix proteins shows these cleavages indeed occur between polar uncharged residues. Previous analysis of these processing sites indicated the importance of positions far from the MIP cleavage site, namely the presence of a hydrophobic residue (Phe or Leu) at P8 and a polar uncharged residue (Ser or Thr) at P5. To evaluate this, additional kinetic analyses were carried out, using fluorogenic substrates synthesized based on the processing sites attributed to MIP. The results described here underscore the importance of the P1 and P1′ substrate positions for the hydrolytic activity of hMIP. The information presented in this work will help in the design of new substrate-based inhibitors for this peptidase. PMID:26082885

A Peptidomic Analysis of Human Milk Digestion in the Infant Stomach Reveals Protein-Specific Degradation Patterns123

PubMed Central

Dallas, David C.; Guerrero, Andrés; Khaldi, Nora; Borghese, Robyn; Bhandari, Aashish; Underwood, Mark A.; Lebrilla, Carlito B.; German, J. Bruce; Barile, Daniela

2014-01-01

In vitro digestion of isolated milk proteins results in milk peptides with a variety of actions. However, it remains unclear to what degree protein degradation occurs in vivo in the infant stomach and whether peptides previously annotated for bioactivity are released. This study combined nanospray LC separation with time-of-flight mass spectrometry, comprehensive structural libraries, and informatics to analyze milk from 3 human mothers and the gastric aspirates from their 4- to 12-d-old postpartum infants. Milk from the mothers contained almost 200 distinct peptides, demonstrating enzymatic degradation of milk proteins beginning either during lactation or between milk collection and feeding. In the gastric samples, 649 milk peptides were identified, demonstrating that digestion continues in the infant stomach. Most peptides in both the intact milk and gastric samples were derived from β-casein. The numbers of peptides from β-casein, lactoferrin, α-lactalbumin, lactadherin, κ-casein, serum albumin, bile salt–associated lipase, and xanthine dehydrogenase/oxidase were significantly higher in the gastric samples than in the milk samples (P < 0.05). A total of 603 peptides differed significantly in abundance between milk and gastric samples (P < 0.05). Most of the identified peptides have previously identified biologic activity. Gastric proteolysis occurs in the term infant in the first 2 wk of life, releasing biologically active milk peptides with immunomodulatory and antibacterial properties of clinical relevance to the proximal intestinal tract. Data are available via ProteomeXchange (identifier PXD000688). PMID:24699806

A peptidomic analysis of human milk digestion in the infant stomach reveals protein-specific degradation patterns.

PubMed

Dallas, David C; Guerrero, Andrés; Khaldi, Nora; Borghese, Robyn; Bhandari, Aashish; Underwood, Mark A; Lebrilla, Carlito B; German, J Bruce; Barile, Daniela

2014-06-01

In vitro digestion of isolated milk proteins results in milk peptides with a variety of actions. However, it remains unclear to what degree protein degradation occurs in vivo in the infant stomach and whether peptides previously annotated for bioactivity are released. This study combined nanospray LC separation with time-of-flight mass spectrometry, comprehensive structural libraries, and informatics to analyze milk from 3 human mothers and the gastric aspirates from their 4- to 12-d-old postpartum infants. Milk from the mothers contained almost 200 distinct peptides, demonstrating enzymatic degradation of milk proteins beginning either during lactation or between milk collection and feeding. In the gastric samples, 649 milk peptides were identified, demonstrating that digestion continues in the infant stomach. Most peptides in both the intact milk and gastric samples were derived from β-casein. The numbers of peptides from β-casein, lactoferrin, α-lactalbumin, lactadherin, κ-casein, serum albumin, bile salt-associated lipase, and xanthine dehydrogenase/oxidase were significantly higher in the gastric samples than in the milk samples (P < 0.05). A total of 603 peptides differed significantly in abundance between milk and gastric samples (P < 0.05). Most of the identified peptides have previously identified biologic activity. Gastric proteolysis occurs in the term infant in the first 2 wk of life, releasing biologically active milk peptides with immunomodulatory and antibacterial properties of clinical relevance to the proximal intestinal tract. Data are available via ProteomeXchange (identifier PXD000688). © 2014 American Society for Nutrition.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wenhui; Chen, Xilei; Li, Tao

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 ligandmore » 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.« less

Designing Authentic Learning Tasks for Online Library Instruction

ERIC Educational Resources Information Center

Finch, Jannette L.; Jefferson, Renee N.

2013-01-01

This empirical study explores whether authentic tasks designed specifically for deliberately grouped students have an effect on student perception of teaching presence and student cognitive gains. In one library research class offered in an express session online, the instructor grouped students randomly. In a second online library research class,…

Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design.

PubMed

Kato, Ryuji; Kaga, Chiaki; Kunimatsu, Mitoshi; Kobayashi, Takeshi; Honda, Hiroyuki

2006-06-01

Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have less influence on the peptide design. Such rules that are indicative of the nature of the functional peptide sequence can be obtained only by the mass comparison analysis of PIASPAC using peptide array. By following such indicative rules, numerous amino acid combinations can be effectively screened for further examination of novel peptide design.

Latin Hypercube Sampling (LHS) UNIX Library/Standalone

DOE Office of Scientific and Technical Information (OSTI.GOV)

2004-05-13

The LHS UNIX Library/Standalone software provides the capability to draw random samples from over 30 distribution types. It performs the sampling by a stratified sampling method called Latin Hypercube Sampling (LHS). Multiple distributions can be sampled simultaneously, with user-specified correlations amongst the input distributions, LHS UNIX Library/ Standalone provides a way to generate multi-variate samples. The LHS samples can be generated either as a callable library (e.g., from within the DAKOTA software framework) or as a standalone capability. LHS UNIX Library/Standalone uses the Latin Hypercube Sampling method (LHS) to generate samples. LHS is a constrained Monte Carlo sampling scheme. Inmore » LHS, the range of each variable is divided into non-overlapping intervals on the basis of equal probability. A sample is selected at random with respect to the probability density in each interval, If multiple variables are sampled simultaneously, then values obtained for each are paired in a random manner with the n values of the other variables. In some cases, the pairing is restricted to obtain specified correlations amongst the input variables. Many simulation codes have input parameters that are uncertain and can be specified by a distribution, To perform uncertainty analysis and sensitivity analysis, random values are drawn from the input parameter distributions, and the simulation is run with these values to obtain output values. If this is done repeatedly, with many input samples drawn, one can build up a distribution of the output as well as examine correlations between input and output variables.« less

Dermatoxin and phylloxin from the waxy monkey frog, Phyllomedusa sauvagei: cloning of precursor cDNAs and structural characterization from lyophilized skin secretion.

PubMed

Chen, Tianbao; Walker, Brian; Zhou, Mei; Shaw, Chris

2005-07-15

Amphibian skin is a morphologically, biochemically and physiologically complex organ that performs the wide range of functions necessary for amphibian survival. Here we describe the primary structures of representatives of two novel classes of amphibian skin antimicrobials, dermatoxin and phylloxin, from the skin secretion of Phyllomedusa sauvagei, deduced from their respective precursor encoding cDNAs cloned from a lyophilized skin secretion library. A degenerate primer, designed to a highly conserved domain in the 5'-untranslated region of analogous peptide precursor cDNAs from Phyllomedusa bicolor, was employed in a 3'-RACE reaction. Peptides with molecular masses coincident with precursor-deduced mature toxin peptides were identified in LC/MS fractions of skin secretion and primary structures were confirmed by MS/MS fragmentation. This integrated experimental approach can thus rapidly expedite the primary structural characterization of amphibian skin peptides in a manner that circumvents specimen sacrifice whilst preserving robustness of scientific data.