Detection of peptides lies at the core of bottom-up proteomics analyses. We examined a Bayesian approach to peptide detection, integrating match-based models (fragments, retention time, isotopic distribution, and precursor mass) and peptide prior probability models under a unified probabilistic framework. To assess the relevance of these models and their various combinations, we employed a complete- and a tail-complete search of a low-precursor-mass synthetic peptide library based on oncogenic KRAS peptides. The fragment match was by far the most informative match-based model, while the retention time match was the only remaining such model with an appreciable impact--increasing correct detections by around 8 %. A peptide prior probability model built from a reference proteome greatly improved the detection over a uniform prior, essentially transforming de novo sequencing into a reference-guided search. The knowledge of a correct sequence tag in advance to peptide-spectrum matching had only a moderate impact on peptide detection unless the tag was long and of high certainty. The approach also derived more precise error rates on the analyzed combinatorial peptide library than those estimated using PeptideProphet and Percolator, showing its potential applicability for the detection of homologous peptides. Although the approach requires further computational developments for routine data analysis, it illustrates the value of peptide prior probabilities and presents a Bayesian approach for their incorporation into peptide detection.
- MeSH
- Algorithms MeSH
- Bayes Theorem MeSH
- Databases, Protein MeSH
- Peptide Library * MeSH
- Peptides * analysis MeSH
- Proteome analysis MeSH
- Proteomics MeSH
- Publication type
- Journal Article MeSH
Described is a computer-assisted rational design of a DNA-bis-intercalator peptide library. The peptide library of 250 members was prepared and the most powerful binder identified. A value of the binding constant is almost two orders of magnitude higher than that of starting building block-9-aminoacridine. The binder affinity found toward calf thymus DNA is 30-fold of that of human prion peptide 106-126.
- MeSH
- Aminacrine chemistry MeSH
- Computer-Aided Design MeSH
- DNA-Binding Proteins chemistry metabolism MeSH
- DNA chemical synthesis chemistry metabolism MeSH
- Financing, Organized MeSH
- Fluorescent Dyes chemistry MeSH
- Gene Library MeSH
- Intercalating Agents chemistry MeSH
- Humans MeSH
- Models, Molecular MeSH
- Peptide Fragments metabolism MeSH
- Prions metabolism MeSH
- Cattle MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Combinatorial Chemistry Techniques MeSH
- Thymus Gland chemistry MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Cattle MeSH
- Animals MeSH
Single-chain antibodies (scFv) exhibiting specific binding to Lawsonia intracellularis were isolated from a phagemid library expressing scFvs molecules on the surface of filamentous bacteriophages. For scFv selection whole bacterial cells were used and individual clones were tested in ELISA test. The total of seven unique clones with different fingerprint profiles was isolated. All clones were able to bind specifically in immunofluorescence assay. This is the first report of species specific recombinant antibodies against L. intracellularis.
We designed a combinatorial library of trifunctional scaffold-derived compounds, which were derivatized with 30 different in-house-made azides. The compounds were proposed to mimic insulin receptor (IR)-binding epitopes in the insulin molecule and bind to and activate this receptor. This work has enabled us to test our synthetic and biological methodology and to prove its robustness and reliability for the solid-phase synthesis and testing of combinatorial libraries of the trifunctional scaffold-derived compounds. Our effort resulted in the discovery of two compounds, which were able to weakly induce the autophosphorylation of IR and weakly bind to this receptor at a 0.1 mM concentration. Despite these modest biological results, which well document the well-known difficulty in modulating protein-protein interactions, this study represents a unique example of targeting the IR with a set of nonpeptide compounds that were specifically designed and synthesized for this purpose. We believe that this work can open new perspectives for the development of next-generation insulin mimetics based on the scaffold structure.
- MeSH
- Azides chemical synthesis chemistry MeSH
- Insulin analogs & derivatives chemistry metabolism MeSH
- Small Molecule Libraries chemical synthesis chemistry metabolism pharmacology MeSH
- Copper analysis MeSH
- Molecular Structure MeSH
- Receptor, Insulin chemistry metabolism MeSH
- Reproducibility of Results MeSH
- Combinatorial Chemistry Techniques * MeSH
- Solid-Phase Synthesis Techniques MeSH
- Protein Binding MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
An efficient and high-yielding solid phase synthesis of a small library of imidazolidin-2-ones and imidazol-2-ones was carried out employing a high chemo- and regioselective gold-catalyzed cycloisomerization as a key step. Polymer-supported amino acids derivatized with several alkyne functionalities combined with tosyl- and phenylureas have been subjected to gold-catalysis exhibiting exclusively C-N bond formation. The present work proves the potential of solid phase synthesis and homogeneous gold catalysis as an efficient and powerful synthetic tool for the generation of drug-like heterocycles.
- MeSH
- Alkynes chemistry MeSH
- Cyclization MeSH
- Imidazolidines chemical synthesis MeSH
- Catalysis MeSH
- Small Molecule Libraries chemical synthesis MeSH
- Molecular Structure MeSH
- Combinatorial Chemistry Techniques MeSH
- Solid-Phase Synthesis Techniques MeSH
- Gold chemistry MeSH
- Publication type
- Journal Article MeSH
The development of canine immunotolerant monoclonal antibodies can accelerate the invention of new medicines for both canine and human diseases. We develop a methodology to clone the naive, somatically mutated variable domain repertoire from canine B cell mRNA using 5'RACE PCR. A set of degenerate primers were then designed and used to clone variable domain genes into archival "holding" plasmid libraries. These archived variable domain genes were then combinatorially ligated to produce a scFv M13 phage library. Next-generation long-read and short-read DNA sequencing methodologies were developed to annotate features of the cloned library including CDR diversity and IGHV/IGKV/IGLV subfamily distribution. A synthetic immunoglobulin G was developed from this scFv library to the canine immune checkpoint receptor PD-1. This synthetic platform can be used to clone and annotate archived antibody variable domain genes for use in perpetuity in order to develop improved preclinical models for the treatment of complex human diseases.
- MeSH
- Programmed Cell Death 1 Receptor immunology MeSH
- Single-Chain Antibodies * immunology genetics MeSH
- Humans MeSH
- Antibodies, Monoclonal immunology genetics MeSH
- Neoplasms immunology therapy MeSH
- Peptide Library MeSH
- Dogs MeSH
- Recombinant Proteins immunology genetics MeSH
- Translational Research, Biomedical MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Dogs MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Defining dynamic protein-protein interactions in the ubiquitin conjugation reaction is a challenging research area. Generating peptide aptamers that target components such as ubiquitin itself, E1, E2, or E3 could provide tools to dissect novel features of the enzymatic cascade. Next-generation deep sequencing platforms were used to identify peptide sequences isolated from phage-peptide libraries screened against Ubiquitin and its ortholog NEDD8. In over three rounds of selection under differing wash criteria, over 13,000 peptides were acquired targeting ubiquitin, while over 10,000 peptides were selected against NEDD8. The overlap in peptides against these two proteins was less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8 toward linear peptide motifs. Two of these ubiquitin-binding peptides were identified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysis highlighted distinct modes of binding of the two different peptide aptamers. These data highlight the utility of using next-generation sequencing of combinatorial phage-peptide libraries to isolate peptide aptamers toward a protein target that can be used as a chemical tool in a complex multi-enzyme reaction.
- Publication type
- Journal Article MeSH
XV, 526 s. : bar.fot., obr., tab., grafy ; 32 cm
- MeSH
- Molecular Biology MeSH
- Publication type
- Encyclopedia MeSH
- Conspectus
- Biologické vědy
- NML Fields
- biologie
- biologie
Galectin-3 (Gal-3), a member of the β-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcβ1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.
- MeSH
- Amino Sugars chemical synthesis chemistry metabolism MeSH
- Galectin 3 antagonists & inhibitors metabolism MeSH
- Glycoproteins chemical synthesis chemistry metabolism pharmacology MeSH
- Glycosylation MeSH
- Humans MeSH
- Ligands MeSH
- Oligosaccharides chemical synthesis chemistry metabolism MeSH
- Serum Albumin, Bovine chemical synthesis chemistry metabolism pharmacology MeSH
- Cattle MeSH
- Combinatorial Chemistry Techniques MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
