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
• algoritmy MeSH
• Bayesova věta MeSH
• databáze proteinů MeSH
• peptidová knihovna * MeSH
• peptidy * analýza   MeSH
• proteom analýza   MeSH
• proteomika MeSH
• Publikační typ
• časopisecké články 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.

• MeSH
• financování organizované MeSH
• Lawsonia (bakterie) imunologie   MeSH
• lidé MeSH
• peptidová knihovna MeSH
• protilátky bakteriální genetika   imunologie   izolace a purifikace   MeSH
• specificita protilátek MeSH
• variabilní oblast imunoglobulinu genetika   imunologie   izolace a purifikace   MeSH
• Check Tag
• lidé 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
• antigeny CD279 imunologie   MeSH
• jednořetězcové protilátky * imunologie   genetika   MeSH
• lidé MeSH
• monoklonální protilátky imunologie   genetika   MeSH
• nádory imunologie   terapie   MeSH
• peptidová knihovna MeSH
• psi MeSH
• rekombinantní proteiny imunologie   genetika   MeSH
• translační biomedicínský výzkum MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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
• aminakrin chemie   MeSH
• design s pomocí počítače MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA chemická syntéza   chemie   metabolismus   MeSH
• financování organizované MeSH
• fluorescenční barviva chemie   MeSH
• genová knihovna MeSH
• interkalátory chemie   MeSH
• lidé MeSH
• molekulární modely MeSH
• peptidové fragmenty metabolismus   MeSH
• priony metabolismus   MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• techniky kombinatorické chemie MeSH
• thymus chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata 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.

• Publikační typ
• časopisecké články MeSH

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic hydrolase acting mostly on nonhistone protein substrates. Investigations of the substrate specificity of HDAC6 are confounded by the presence of 2 catalytically active deacetylase domains (DD1 and DD2). In this study, acetylome peptide microarrays and peptide libraries were used to map the substrate specificity of DD1 and DD2 of human HDAC6. The results show that DD1 is solely responsible for the deacetylation of substrates harboring the acetyllysine at their C terminus, whereas DD2 exclusively deacetylates peptides with an internal acetyllysine residue. Also, statistical analysis of the deacetylation data revealed amino acid preferences at individual positions flanking the acetyllysine, where glycine and arginine residues are favored at positions N-terminal to the central acetyllysine; negatively charged glutamate is strongly disfavored throughout the sequence. Finally, the deacylation activity of HDAC6 was profiled by using a panel of acyl derivatives of the optimized peptide substrate and showed that HDAC6 acts as a proficient deformylase. Our data thus offer a detailed insight into the substrate preferences of the individual HDAC6 domains at the peptide level, and these findings can in turn help in elucidating the biologic roles of the enzyme and facilitate the development of new domain-specific inhibitors as research tools or therapeutic agents.-Kutil, Z., Skultetyova, L., Rauh, D., Meleshin, M., Snajdr, I., Novakova, Z., Mikesova, J., Pavlicek, J., Hadzima, M., Baranova, P., Havlinova, B., Majer, P., Schutkowski, M., Barinka, C. The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.

• MeSH
• HEK293 buňky MeSH
• histondeacetylasa 6 chemie   metabolismus   MeSH
• katalytická doména * MeSH
• lidé MeSH
• lysin chemie   metabolismus   MeSH
• peptidové fragmenty chemie   metabolismus   MeSH
• statická elektřina MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Trichobilharzia regenti is a neurotropic bird schistosome,causing cercarial dermatitis in humans. In this study, ZAP cDNA expression library from Radix peregra s. lat. hepatopancreases containing intramolluscan stages of T. regenti was constructed and screened using PCR with specific and degenerate primers, designed according to previously described serine and cysteine peptidases of other parasite species. Full-length sequences of cathepsins B1 and L, and two serine peptidases, named RpSP1 and RpSP2, were obtained. The protein-protein BLAST analysis and parallel control reactions with template from hepatopancreases of T. regenti non-infected snails revealed that only cathepsin B1 was of parasite origin. The remaining sequences were derived from the snail intermediate host, which implies that the initial source of parasite mRNA was contaminated by snail tissue. Regardless of this contamination, the cDNA library remains an excellent molecular tool for detection and identification of bioactive molecules in T. regenti cercariae.

• MeSH
• cysteinové endopeptidasy genetika   MeSH
• genová knihovna MeSH
• hlemýždi parazitologie   MeSH
• kathepsin B genetika   MeSH
• kathepsin L MeSH
• kathepsiny genetika   MeSH
• molekulární sekvence - údaje MeSH
• polymerázová řetězová reakce MeSH
• proteasy genetika   MeSH
• proteiny červů genetika   MeSH
• Schistosomatidae enzymologie   genetika   růst a vývoj   MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie aminokyselin MeSH
• stadia vývoje MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• molekulární biologie MeSH
• Publikační typ
• encyklopedie MeSH

• Konspekt
• Biologické vědy
• NLK Obory
• biologie
• biologie

HotSpot Wizard 2.0 is a web server for automated identification of hot spots and design of smart libraries for engineering proteins' stability, catalytic activity, substrate specificity and enantioselectivity. The server integrates sequence, structural and evolutionary information obtained from 3 databases and 20 computational tools. Users are guided through the processes of selecting hot spots using four different protein engineering strategies and optimizing the resulting library's size by narrowing down a set of substitutions at individual randomized positions. The only required input is a query protein structure. The results of the calculations are mapped onto the protein's structure and visualized with a JSmol applet. HotSpot Wizard lists annotated residues suitable for mutagenesis and can automatically design appropriate codons for each implemented strategy. Overall, HotSpot Wizard provides comprehensive annotations of protein structures and assists protein engineers with the rational design of site-specific mutations and focused libraries. It is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard.

• MeSH
• automatizace MeSH
• biokatalýza MeSH
• databáze proteinů MeSH
• internet * MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• mutace * MeSH
• mutageneze cílená metody   MeSH
• peptidová knihovna * MeSH
• proteiny chemie   genetika   MeSH
• software * MeSH
• stabilita proteinů MeSH
• substituce aminokyselin MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH

DNA sequences offer powerful tools for describing the members and interactions of natural communities. In this study, we establish the to-date most comprehensive library of DNA barcodes for a terrestrial site, including all known macroscopic animals and vascular plants of an intensively studied area of the High Arctic, the Zackenberg Valley in Northeast Greenland. To demonstrate its utility, we apply the library to identify nearly 20 000 arthropod individuals from two Malaise traps, each operated for two summers. Drawing on this material, we estimate the coverage of previous morphology-based species inventories, derive a snapshot of faunal turnover in space and time and describe the abundance and phenology of species in the rapidly changing arctic environment. Overall, 403 terrestrial animal and 160 vascular plant species were recorded by morphology-based techniques. DNA barcodes (CO1) offered high resolution in discriminating among the local animal taxa, with 92% of morphologically distinguishable taxa assigned to unique Barcode Index Numbers (BINs) and 93% to monophyletic clusters. For vascular plants, resolution was lower, with 54% of species forming monophyletic clusters based on barcode regions rbcLa and ITS2. Malaise catches revealed 122 BINs not detected by previous sampling and DNA barcoding. The insect community was dominated by a few highly abundant taxa. Even closely related taxa differed in phenology, emphasizing the need for species-level resolution when describing ongoing shifts in arctic communities and ecosystems. The DNA barcode library now established for Zackenberg offers new scope for such explorations, and for the detailed dissection of interspecific interactions throughout the community.

• MeSH
• ekosystém * MeSH
• fylogeneze MeSH
• mezerníky ribozomální DNA chemie   genetika   MeSH
• respirační komplex IV genetika   MeSH
• ribulosa-1,5-bisfosfát-karboxylasa genetika   MeSH
• rostliny MeSH
• společenstvo * MeSH
• taxonomické DNA čárové kódování metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Arktida MeSH
• Grónsko MeSH