The Experts below are selected from a list of 105081 Experts worldwide ranked by ideXlab platform
Sergey Rodin - One of the best experts on this subject based on the ideXlab platform.
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system wide identification and prioritization of Enzyme Substrates by thermal analysis
Nature Communications, 2021Co-Authors: Amir Ata Saei, Christian M Beusch, Pierre Sabatier, Juan Astorga Wells, Hassan Gharibi, Zhaowei Meng, Alexey Chernobrovkin, Sergey RodinAbstract:Despite the immense importance of Enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the Enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of Enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery. The global identification of Enzyme Substrates is still challenging. Here, the authors develop a method based on proteome-wide thermal shift assays to discover Enzyme Substrates directly from cell lysates, identifying known and novel oxidoreductase, kinase and poly-(ADP-ribose) polymerase Substrates.
Amir Ata Saei - One of the best experts on this subject based on the ideXlab platform.
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system wide identification and prioritization of Enzyme Substrates by thermal analysis
Nature Communications, 2021Co-Authors: Amir Ata Saei, Christian M Beusch, Pierre Sabatier, Juan Astorga Wells, Hassan Gharibi, Zhaowei Meng, Alexey Chernobrovkin, Sergey RodinAbstract:Despite the immense importance of Enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the Enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of Enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery. The global identification of Enzyme Substrates is still challenging. Here, the authors develop a method based on proteome-wide thermal shift assays to discover Enzyme Substrates directly from cell lysates, identifying known and novel oxidoreductase, kinase and poly-(ADP-ribose) polymerase Substrates.
Sergey A. Piletsky - One of the best experts on this subject based on the ideXlab platform.
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Highly Efficient Abiotic Assay Formats for Methyl Parathion: Molecularly Imprinted Polymer Nanoparticle Assay as an Alternative to Enzyme-Linked Immunosorbent Assay
2018Co-Authors: Cem Esen, Joanna Czulak, Todd Cowen, Elena Piletska, Sergey A. PiletskyAbstract:Enzyme-linked immunosorbent assay (ELISA) is a widely used standard method for sensitive detection of analytes of environmental, clinical, or biotechnological interest. However, ELISA has clear drawbacks related to the use of relatively unstable antibodies and Enzyme conjugates and the need for several steps such as washing of nonbound conjugates and addition of dye reagents. Herein, we introduce a new completely abiotic assay where antibodies and Enzymes are replaced with fluorescent molecularly imprinted polymer nanoparticles (nanoMIPs) and target-conjugated magnetic nanoparticles, which acted as both reporter probes and binding agents. The components of the molecularly imprinted polymer nanoparticle assay (MINA) are assembled in microtiter plates fitted with magnetic inserts. We have compared the performance of a new magnetic assay with molecularly imprinted polymer (MIP)-based ELISA for the detection of methyl parathion (MP). Both assays have shown high sensitivity toward allowing detection of MP at picomolar concentrations without any cross-reactivity against chlorpyriphos and fenthion. The fully abiotic assays were also proven to detect analyte in real samples such as tap water and milk. Unlike ELISA-based systems, the novel assay required no washing steps or addition of Enzyme Substrates, making it more user-friendly and suitable for high throughput screening
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Highly Efficient Abiotic Assay Formats for Methyl Parathion: Molecularly Imprinted Polymer Nanoparticle Assay as an Alternative to Enzyme-Linked Immunosorbent Assay
2018Co-Authors: Cem Esen, Joanna Czulak, Todd Cowen, Elena Piletska, Sergey A. PiletskyAbstract:Enzyme-linked immunosorbent assay (ELISA) is a widely used standard method for sensitive detection of analytes of environmental, clinical, or biotechnological interest. However, ELISA has clear drawbacks related to the use of relatively unstable antibodies and Enzyme conjugates and the need for several steps such as washing of nonbound conjugates and addition of dye reagents. Herein, we introduce a new completely abiotic assay where antibodies and Enzymes are replaced with fluorescent molecularly imprinted polymer nanoparticles (nanoMIPs) and target-conjugated magnetic nanoparticles, which acted as both reporter probes and binding agents. The components of the molecularly imprinted polymer nanoparticle assay (MINA) are assembled in microtiter plates fitted with magnetic inserts. We have compared the performance of a new magnetic assay with molecularly imprinted polymer (MIP)-based ELISA for the detection of methyl parathion (MP). Both assays have shown high sensitivity toward allowing detection of MP at picomolar concentrations without any cross-reactivity against chlorpyriphos and fenthion. The fully abiotic assays were also proven to detect analyte in real samples such as tap water and milk. Unlike ELISA-based systems, the novel assay required no washing steps or addition of Enzyme Substrates, making it more user-friendly and suitable for high throughput screening
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A Novel Assay Format as an Alternative to ELISA: MINA Test for Biotin
2018Co-Authors: Joanna Czulak, Elena Piletska, Aeg Cass, Sergey A. PiletskyAbstract:A novel abiotic assay based on biotin‐specific fluorescent molecularly imprinted polymer nanoparticles (nanoMIPs) which acted as both reporter probes and binding agents, was developed. This is a first report of an assay which, unlike ELISA, required no washing steps or addition of Enzyme Substrates, making it more user‐friendly. The components of the molecularly imprinted polymer nanoparticles assay (MINA) were assembled in microtiter plates fitted with magnetic inserts. The fluorescent nanoMIPs were bound to biotin‐conjugated magnetic particles, which were attracted to the inserts. The addition of free biotin caused a displacement of the fluorescent nanoMIPs into solution, generating a signal proportional to the concentration of biotin. The nanoMIPs had a dissociation constant (Kd) of 14 nM, allowing the assay to detect biotin at nano‐molar concentrations. The pre‐assembled assay only required the addition of the sample and measurement of the fluorescence, and it functioned well after six weeks of storage without refrigeration. The assay did not show the susceptibility to several compounds which are known to interfere with avidin and streptavidin‐based assays, such as mercaptoethanol and sugars. The protocols optimized in this work could be used to develop the abiotic assays for any other compound of interest
A. Tatibouët - One of the best experts on this subject based on the ideXlab platform.
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Bifunctional mannoside-glucosinolate glycoconjugates as enzymatically triggered isothiocyanates and FimH ligands
Organic and Biomolecular Chemistry, 2018Co-Authors: G. Cutolo, F. Reise, M. Schuler, R. Nehmé, G. Despras, J. Brekalo, P. Morin, Pierre-yves Renard, T.k. Lindhorst, A. TatibouëtAbstract:Glucosinolates are sulfur-containing secondary metabolites found in plants of the Brassicale order. They are precursors of isothiocyanate species, resulting from C–S hydrolysis catalysed by the thioglucohydrolase myrosinase. We describe the synthesis of bifunctional glucosinolate–mannoside glycoconjugates combining both the structural features of a substrate of myrosinase and a ligand of the lectin FimH. We show that these glycoconjugates serve as Enzyme Substrates and that myrosinase can indeed hydrolyze the glucosinolate moiety with affinities (KM, Vmax) comparable to the natural Substrates glucomoringin and sinigrin. This enzymatic hydrolysis of the thioglycosidic bond led to the efficient formation of an isothiocyanate which was assessed by the formation of the corresponding dithiocarbamate derivatives. Finally, we show that our synthetic bifunctional glycoconjugates also serve as FimH ligands where the glucosinolate moiety does not hamper the interaction with the lectin. Our findings set the stage for an original bioconjugation tool, allowing for myrosinase-triggered specific labelling of lectins using glucosinolate glycoconjugates as non-toxic, water soluble isothiocyanate precursors.
John R. Dean - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Listeria using exogenous volatile organic compound metabolites and their detection by static headspace–multi-capillary column–gas chromatography–ion mobility spectrometry (SHS–MCC–GC–IMS)
Analytical and Bioanalytical Chemistry, 2017Co-Authors: Carl Taylor, Fraser Lough, Ian A. Fowlis, Edward C Schwalbe, S.p. Stanforth, John R. DeanAbstract:Listeria monocytogenes is a Gram-positive bacterium and an opportunistic food-borne pathogen which poses significant risk to the immune-compromised and pregnant due to the increased likelihood of acquiring infection and potential transmission of infection to the unborn child. Conventional methods of analysis suffer from either long turn-around times or lack the ability to discriminate between Listeria spp. reliably. This paper investigates an alternative method of detecting Listeria spp. using two novel Enzyme Substrates that liberate exogenous volatile organic compounds in the presence of α-mannosidase and d -alanyl aminopeptidase. The discriminating capabilities of this approach for identifying L. monocytogenes from other species of Listeria are investigated. The liberated volatile organic compounds (VOCs) are detected using an automated analytical technique based on static headspace–multi-capillary column–gas chromatography–ion mobility spectrometry (SHS–MCC–GC–IMS). The results obtained by SHS–MCC–GC–IMS are compared with those obtained by the more conventional analytical technique of headspace–solid phase microextraction–gas chromatography–mass spectrometry (HS–SPME–GC–MS). The results found that it was possible to differentiate between L. monocytogenes and L. ivanovii , based on their VOC response from α-mannosidase activity.
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detection of exogenous vocs as a novel in vitro diagnostic technique for the detection of pathogenic bacteria
Trends in Analytical Chemistry, 2017Co-Authors: Fraser Lough, John D Perry, Stephen P Stanforth, John R. DeanAbstract:The evolution of volatile organic compounds (VOCs) provides an opportunistic approach for the detection of pathogenic bacteria. This approach can be enhanced by the application of VOC-labelled Enzyme Substrates that target specific Enzyme activities of the bacteria under investigation. Detection of the VOCs provides a novel, specific and sensitive approach for the detection of pathogenic bacteria. This review highlights the importance of this approach alongside a range of alternative detection techniques for the identification of pathogenic bacteria.
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bacteria detection based on the evolution of Enzyme generated volatile organic compounds determination of listeria monocytogenes in milk samples
Analytica Chimica Acta, 2014Co-Authors: Emma Tait, Stephen P Stanforth, John D Perry, John R. DeanAbstract:The rapid detection of Listeria monocytogenes contamination in food is essential to prevent food-borne illness in humans. The aim of this study was to differentiate non-contaminated milk from milk contaminated with L. monocytogenes using Enzyme Substrates coupled with the analysis of volatile organic compounds (VOCs). The method is based on the activity of β-glucosidase and hippuricase Enzymes and the detection of a specific VOC i.e. 2-nitrophenol and 3-fluoroaniline, respectively. VOCs were extracted, separated and detected by headspace-solid phase microextraction coupled to gas chromatography–mass spectrometry (HS-SPME GC–MS). This approach required the inclusion of the selective agent's cycloheximide, nalidixic acid and acriflavine HCl in the growth medium to inhibit interfering bacteria. The VOCs were liberated by L. monocytogenes provided that samples contained at least 1–1.5 × 102 CFU ml−1 of milk prior to overnight incubation. This approach shows potential for future development as a rapid method for the detection of L. monocytogenes contaminated milk.