A High-Throughput, Low-Volume Enzyme Assay on Solid Support

Babiak, Peter; Reymond, Jean‐Louis

doi:10.1021/ac048611n

Cited by 63 publications

(40 citation statements)

References 10 publications

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“…87 Alternative surfaces have further been developed for droplet-based enzyme profiling. [88][89][90] Using slides coated with fluorogenic substrates, activity-dependent profiles of proteases and phosphates may obtained in nanoliter sized droplets, in a time-and concentration-dependent manner. 90 This method was applied to screen for inhibitors of metalloproteases, namely, thermolysin and collagenase, using nearly 150-fold less substrate in comparison to the traditional microplate method.…”

Section: A Substrate Profilingmentioning

confidence: 99%

Microarray-based enzyme profiling: Recent advances and applications (Review)

Uttamchandani

Moochhala

2010

Biointerphases

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Enzymes are an integral part of biological systems. They constitute a significant majority of all proteins expressed (an estimated 18%–29%) within eukaryotic genomes. It thus comes as no major surprise that enzymes have been implicated in many diseases and form the second largest group of drug targets, after receptors. Despite their involvement in a multitude of physiological processes, only a limited number of enzymes have thus far been well-characterized. Consequently, little is understood about the physiological roles, substrate specificity, and downstream targets of the vast majority of these important proteins. In order to facilitate the biological characterization of enzymes, as well as their adoption as drug targets, there is a need for global “-omics” solutions that bridge the gap in understanding these proteins and their interactions. Herein the authors showcase how microarray methods can be adopted to facilitate investigations into enzymes and their properties, in a high-throughput manner. They will focus on several major classes of enzymes, including kinases, phosphatases, and proteases. As a result of research efforts over the last decade, these groups of enzymes have become readily amenable to microarray-based profiling methods. The authors will also describe the specific design considerations that are required to develop the appropriate chemical tools and libraries to characterize each enzyme class. These include peptide substrates, activity-based probes, and chemical compound libraries, which may be rapidly assembled using efficient combinatorial synthesis or “click chemistry” strategies. Taken together, microarrays offer a powerful means to study, profile, and also discover potent small molecules with which to modulate enzyme activity.

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Section: A Substrate Profilingmentioning

confidence: 99%

Microarray-based enzyme profiling: Recent advances and applications (Review)

Uttamchandani

Moochhala

2010

Biointerphases

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“…Because lipases are interfacial enzymes, there has been a search for an assay format where these substrates would be presented to the enzyme solution in stable, insoluble form over a high surface material. The best implementation of this principle consists of impregnating silica gel plates (used for analytical thinlayer chromatography) with a solution of the umbelliferone esters in dichloromethane [28] . Evaporation of the solvent leaves the substrate homogeneously adsorbed on the surface.…”

Section: Assays On Solid Supportmentioning

confidence: 99%

Fluorescence Assays for Biotransformations

Reymond

2008

Modern Biocatalysis

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“…Presently, immobilized enzymes have been widely utilized. Compared with conventional in-solution digestion, immobilized enzymes can greatly shorten the digestion time [25]. Recently, various beads and surface materials have been utilized for protease immobilization [26], among which magnetic microspheres have attracted increasing attention in our group [27][28][29][30][31].…”

Section: Introductionmentioning

confidence: 99%

High Throughput Enzyme Inhibitor Screening by Functionalized Magnetic Carbonaceous Microspheres and Graphene Oxide-Based MALDI-TOF-MS

Liu

et al. 2011

J. Am. Soc. Mass Spectrom.

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In this work, a high throughput methodology for screening enzyme inhibitors has been demonstrated by combining enzyme immobilized magnetic carbonaceous microspheres and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with grapheme oxide as matrix. First, model enzyme acetylcholinesterase (AChE) was immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic carbonaceous (MC) microspheres, displaying a high enzyme activity and stability, and also facilitating the separation of enzyme from substrate and product. The efficiency of immobilized AChE was monitored by biochemical assay, which was carried out by mixing enzyme-immobilized MC microspheres with model substrate acetylcholine (ACh), and subsequent quantitative determination of substrate ACh and product choline using graphene oxide-based MALDI-TOF-MS with no background inference. The limit of detection (LOD) for ACh was 0.25 fmol/μL, and excellent linearity (R 2 =0.9998) was maintained over the range of 0.5 and 250 fmol/μL. Choline was quantified over the range of 0.05 and 15 pmol/μL, also with excellent linearity (R 2 =0.9994) and low LOD (0.15 fmol/μL). Good accuracy and precision were obtained for all concentrations within the range of the standard curves. All together, eight compounds (four known AChE inhibitors and four control chemical compounds with no AChE inhibit effect) were tested with our promoted methodology, and the obtained results demonstrated that our high throughput screening methodology could be a great help to the routine enzyme inhibitor screening.

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A High-Throughput, Low-Volume Enzyme Assay on Solid Support

Cited by 63 publications

References 10 publications

Microarray-based enzyme profiling: Recent advances and applications (Review)

Microarray-based enzyme profiling: Recent advances and applications (Review)

Fluorescence Assays for Biotransformations

High Throughput Enzyme Inhibitor Screening by Functionalized Magnetic Carbonaceous Microspheres and Graphene Oxide-Based MALDI-TOF-MS

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