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Oliver Schilling - One of the best experts on this subject based on the ideXlab platform.
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Profiling of Protease Cleavage Sites by Proteome-Derived Peptide Libraries and Quantitative Proteomics.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Chia-yi Chen, Bettina Mayer, Oliver SchillingAbstract:Biochemical profiling of active site specificity is a crucial step to characterize proteases, which play key roles in health and disease. Here, we present a protocol using proteome-derived Peptide Libraries in combination with quantitative proteomics to simultaneously identify cleavage motifs N- and C-terminal to the scissile Peptide bond. First, bacterial or eukaryotic cell lysate is used to generate Peptide Libraries. Without further chemical modification, Peptide Libraries are then split into control and treated (incubate with active protease) aliquots. Control and treated Libraries are stable isotope-labeled, mixed, and analyzed by liquid chromatography-tandem mass spectrometry. Enriched, semi-specific Peptides represent the cleavage products of the test protease and the entire Peptide sequence that encompasses the scissile Peptide bond is reconstructed bioinformatically. The method is fast, cost-effective, and suited for proteases with narrow or loose specificity.
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characterization of the prime and non prime active site specificities of proteases by proteome derived Peptide Libraries and tandem mass spectrometry
Nature Protocols, 2011Co-Authors: Oliver Schilling, Pitter F Huesgen, Olivier Barre, Ulrich Auf Dem Keller, Christopher M OverallAbstract:Characterization of the prime and non-prime active site specificities of proteases by proteome-derived Peptide Libraries and tandem mass spectrometry
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proteome derived database searchable Peptide Libraries for identifying protease cleavage sites
Nature Biotechnology, 2008Co-Authors: Oliver Schilling, Christopher M OverallAbstract:We introduce human proteome–derived, database-searchable Peptide Libraries for characterizing sequence-specific protein interactions. To identify endoprotease cleavage sites, we used Peptides in such Libraries with protected primary amines to simultaneously determine sequence preferences on the N-terminal (nonprime P) and C-terminal (prime P′) sides of the scissile bond. Prime-side cleavage products were tagged with biotin, isolated and identified by tandem mass spectrometry, and the corresponding nonprime-side sequences were derived from human proteome databases using bioinformatics. Identification of hundreds to over 1,000 individual cleaved Peptides allows the consensus protease cleavage site and subsite cooperativity to be readily determined from P6 to P6′. For the highly specific GluC protease, >95% of the 558 cleavage sites identified displayed the canonical selectivity. For the broad-specificity matrix metalloproteinase 2, >1,200 peptidic cleavage sites were identified. Profiling of HIV protease 1, caspase 3, caspase 7, cathepsins K and G, elastase and thrombin showed that this approach is broadly applicable to all mechanistic classes of endoproteases.
Christopher M Overall - One of the best experts on this subject based on the ideXlab platform.
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cleavage specificity analysis of six type ii transmembrane serine proteases ttsps using pics with proteome derived Peptide Libraries
PLOS ONE, 2014Co-Authors: Olivier Barre, Antoine Dufour, Ulrich Eckhard, Reinhild Kappelhoff, Francois Beliveau, Richard Leduc, Christopher M OverallAbstract:Background Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors. Methodology/Principal Finding To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS). Human proteome-derived database searchable Peptide Libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin) to simultaneously determine sequence preferences on the N-terminal non-prime (P) and C-terminal prime (P’) sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved Peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1′ position. TTSP cleavage between R↓K was confirmed using synthetic Peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived. Conclusions Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1′ positions for arginine and lysine in unfolded Peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.
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characterization of the prime and non prime active site specificities of proteases by proteome derived Peptide Libraries and tandem mass spectrometry
Nature Protocols, 2011Co-Authors: Oliver Schilling, Pitter F Huesgen, Olivier Barre, Ulrich Auf Dem Keller, Christopher M OverallAbstract:Characterization of the prime and non-prime active site specificities of proteases by proteome-derived Peptide Libraries and tandem mass spectrometry
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proteome derived database searchable Peptide Libraries for identifying protease cleavage sites
Nature Biotechnology, 2008Co-Authors: Oliver Schilling, Christopher M OverallAbstract:We introduce human proteome–derived, database-searchable Peptide Libraries for characterizing sequence-specific protein interactions. To identify endoprotease cleavage sites, we used Peptides in such Libraries with protected primary amines to simultaneously determine sequence preferences on the N-terminal (nonprime P) and C-terminal (prime P′) sides of the scissile bond. Prime-side cleavage products were tagged with biotin, isolated and identified by tandem mass spectrometry, and the corresponding nonprime-side sequences were derived from human proteome databases using bioinformatics. Identification of hundreds to over 1,000 individual cleaved Peptides allows the consensus protease cleavage site and subsite cooperativity to be readily determined from P6 to P6′. For the highly specific GluC protease, >95% of the 558 cleavage sites identified displayed the canonical selectivity. For the broad-specificity matrix metalloproteinase 2, >1,200 peptidic cleavage sites were identified. Profiling of HIV protease 1, caspase 3, caspase 7, cathepsins K and G, elastase and thrombin showed that this approach is broadly applicable to all mechanistic classes of endoproteases.
Toshiyuki Mori - One of the best experts on this subject based on the ideXlab platform.
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Human IgA-binding Peptides selected from random Peptide Libraries: Affinity maturation and application in IGA purification
Journal of Biological Chemistry, 2012Co-Authors: Takaaki Hatanaka, Shinji Ohzono, Mirae Park, Shogo Tsukamoto, Ryohei Sugita, Hiroyuki Ishitobi, Kotaro Sakamoto, Osamu Ito, Toshiyuki Mori, Koichi SorajoAbstract:Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random Peptide Libraries constructed with the T7 phage display system to isolate Peptides specific to human immunoglobulin A (IgA). The binding clones (A1-A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic Peptide derived from the A2 clone exhibited a low specificity/affinity (Kd = 1.3 microM). Therefore, we tried to improve the Peptide using a partial randomized phage display library and mutational studies on the synthetic Peptides. The designed Opt-1 Peptide exhibited a 39-fold higher affinity (Kd = 33 nM) than the A2 Peptide. An Opt-1 Peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating non-specific binding. To design a Peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics (MD) simulations with explicit water. The simulation results revealed that the Opt-1 Peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to non-specific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 Peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.
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t7 lytic phage displayed Peptide Libraries exhibit less sequence bias than m13 filamentous phage displayed Peptide Libraries
Proteomics, 2006Co-Authors: Lrh Krumpe, A. Atkinson, Gary W. Smythers, Andrea Kandel, Kathryn M. Schumacher, James B. Mcmahon, Lee Makowski, Toshiyuki MoriAbstract:We investigated whether the T7 system of phage display could produce Peptide Libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random Peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with Peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C Libraries. Based on this analysis, Peptide Libraries constructed with the T7 system have fewer amino acid biases, increased Peptide diversity, and more normal distributions of Peptide net charge and hydropathy than the M13 Libraries. The greater diversity of T7-displayed Libraries provides a potential resource of novel binding Peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed Peptide Libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.
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The use of phage-displayed Peptide Libraries to develop tumor-targeting drugs
International Journal of Peptide Research and Therapeutics, 2006Co-Authors: Lrh Krumpe, Toshiyuki MoriAbstract:Monoclonal antibodies have been successfully utilized as cancer-targeting therapeutics and diagnostics, but the efficacies of these treatments are limited in part by the size of the molecules and non-specific uptake by the reticuloendothelial system. Peptides are much smaller molecules that can specifically target cancer cells and as such may alleviate complications with antibody therapy. Although many endogenous and exogenous Peptides have been developed into clinical therapeutics, only a subset of these consists of cancer-targeting Peptides. Combinatorial biological Libraries such as bacteriophage-displayed Peptide Libraries are a resource of potential ligands for various cancer-related molecular targets. Target-binding Peptides can be affinity selected from complex mixtures of billions of displayed Peptides on phage and further enriched through the biopanning process. Various cancer-specific ligands have been isolated by in vitro, in vivo, and ex vivo screening methods. As several Peptides derived from phage-displayed Peptide library screenings have been developed into therapeutics in current clinical trials, which validates Peptide-targeting potential, the use of phage display to identify cancer-targeting therapeutics should be further exploited.
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T7 lytic phage‐displayed Peptide Libraries exhibit less sequence bias than M13 filamentous phage‐displayed Peptide Libraries
Proteomics, 2006Co-Authors: Lrh Krumpe, A. Atkinson, Gary W. Smythers, Andrea Kandel, Kathryn M. Schumacher, James B. Mcmahon, Lee Makowski, Toshiyuki MoriAbstract:We investigated whether the T7 system of phage display could produce Peptide Libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random Peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with Peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C Libraries. Based on this analysis, Peptide Libraries constructed with the T7 system have fewer amino acid biases, increased Peptide diversity, and more normal distributions of Peptide net charge and hydropathy than the M13 Libraries. The greater diversity of T7-displayed Libraries provides a potential resource of novel binding Peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed Peptide Libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.
Patrick S.c. Leung - One of the best experts on this subject based on the ideXlab platform.
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screening and identification of mimotopes of the major shrimp allergen tropomyosin using one bead one compound Peptide Libraries
Cellular & Molecular Immunology, 2017Co-Authors: Nicki Y. H. Leung, Jinjun Wang, Kit S. Lam, Ruiwu Liu, Christine Yy Wai, Shang An Shu, Ka Hou Chu, Patrick S.c. LeungAbstract:The one-bead-one-compound (OBOC) combinatorial Peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immunoglobulin E (IgE) epitopes of the major shellfish allergen tropomyosin. OBOC Peptide Libraries with 8-12 amino acid residues were screened with serum samples from patients with shellfish allergy for IgE mimotopes of tropomyosin. Twenty-five mimotopes were identified from the screening and their binding reactivity to tropomyosin-specific IgE was confirmed by Peptide ELISA. These mimotopes could be divided into seven clusters based on sequence homology, and epitope mapping by EpiSearch of the clustered mimotopes was performed to characterize and confirm the validity of mimotopes. Five out of six of the predicted epitopes were found to overlap with previously identified epitopes of tropomyosin. To further confirm the mimicry potential of mimotopes, BALB/c mice were immunized with mimotopes conjugated to keyhole limpet hemocyanin and assayed for their capacity to induce tropomyosin-specific antibodies. BALB/c mice that received mimotope immunization were found to have an elevated level of tropomyosin-specific immunoglobulin G, but not mice that received an irrelevant mimotope. This study pioneers the successful application of the OBOC Libraries using whole sera to screen and identify multiple shrimp allergen mimotopes and validates their mimicry potential using in vitro, in vivo, and in silico methods.Cellular & Molecular Immunology advance online publication, 14 september 2015; doi:10.1038/cmi.2015.83.
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Screening and identification of mimotopes of the major shrimp allergen tropomyosin using one-bead-one-compound Peptide Libraries.
Cellular & Molecular Immunology, 2015Co-Authors: Nicki Y. H. Leung, Marco Hk Ho, Jinjun Wang, Patrick S.c. LeungAbstract:The one-bead-one-compound (OBOC) combinatorial Peptide library is a powerful tool to identify ligand and receptor interactions. Here, we applied the OBOC library technology to identify mimotopes specific to the immunoglobulin E (IgE) epitopes of the major shellfish allergen tropomyosin. OBOC Peptide Libraries with 8–12 amino acid residues were screened with serum samples from patients with shellfish allergy for IgE mimotopes of tropomyosin. Twenty-five mimotopes were identified from the screening and their binding reactivity to tropomyosin-specific IgE was confirmed by Peptide ELISA. These mimotopes could be divided into seven clusters based on sequence homology, and epitope mapping by EpiSearch of the clustered mimotopes was performed to characterize and confirm the validity of mimotopes. Five out of six of the predicted epitopes were found to overlap with previously identified epitopes of tropomyosin. To further confirm the mimicry potential of mimotopes, BALB/c mice were immunized with mimotopes conjugated to keyhole limpet hemocyanin and assayed for their capacity to induce tropomyosin-specific antibodies. BALB/c mice that received mimotope immunization were found to have an elevated level of tropomyosin-specific immunoglobulin G, but not mice that received an irrelevant mimotope. This study pioneers the successful application of the OBOC Libraries using whole sera to screen and identify multiple shrimp allergen mimotopes and validates their mimicry potential using in vitro, in vivo, and in silico methods.
Martin Trepel - One of the best experts on this subject based on the ideXlab platform.
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Biomathematical description of synthetic Peptide Libraries.
PloS one, 2015Co-Authors: Timo Sieber, Eric Hare, Heike Hofmann, Martin TrepelAbstract: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.
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Novel random Peptide Libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors
Gene Therapy, 2012Co-Authors: Kata Váradi, Jürgen A. Kleinschmidt, Stefan Michelfelder, Martin Trepel, Hugo A Katus, Matthias Hecker, Thomas Korff, Oliver J. MüllerAbstract:We have demonstrated the potential of random Peptide Libraries displayed on adeno-associated virus (AAV)2 to select for AAV2 vectors with improved efficiency for cell type-directed gene transfer. AAV9, however, may have advantages over AAV2 because of a lower prevalence of neutralizing antibodies in humans and more efficient gene transfer in vivo . Here we provide evidence that random Peptide Libraries can be displayed on AAV9 and can be utilized to select for AAV9 capsids redirected to the cell type of interest. We generated an AAV9 Peptide display library, which ensures that the displayed Peptides correspond to the packaged genomes and performed four consecutive selection rounds on human coronary artery endothelial cells in vitro . This screening yielded AAV9 library capsids with distinct Peptide motifs enabling up to 40-fold improved transduction efficiencies compared with wild-type (wt) AAV9 vectors. Incorporating sequences selected from AAV9 Libraries into AAV2 capsids could not increase transduction as efficiently as in the AAV9 context. To analyze the potential on endothelial cells in the intact natural vascular context, human umbilical veins were incubated with the selected AAV in situ and endothelial cells were isolated. Fluorescence-activated cell sorting analysis revealed a 200-fold improved transduction efficiency compared with wt AAV9 vectors. Furthermore, AAV9 vectors with targeting sequences selected from AAV9 Libraries revealed an increased transduction efficiency in the presence of human intravenous immunoglobulins, suggesting a reduced immunogenicity. We conclude that our novel AAV9 Peptide library is functional and can be used to select for vectors for future preclinical and clinical gene transfer applications.
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successful expansion but not complete restriction of tropism of adeno associated virus by in vivo biopanning of random virus display Peptide Libraries
PLOS ONE, 2009Co-Authors: Stefan Michelfelder, Jürgen A. Kleinschmidt, Oliver J. Müller, Martin Trepel, Johannes Kohlschutter, Alexandra Skorupa, Sabrina PfenningsAbstract:Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random Peptide Libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for Peptide Libraries displayed on adeno-associated virus (AAV), we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV Peptide Libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of Peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding Peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical.