Based on the FPIA of high-throughput gene and protein detection analysis system

2008 ◽  
pp. 404-407
Author(s):  
Ni Yuan ◽  
Ju Zhang ◽  
Lihong Yang ◽  
Yanhai Guo ◽  
Zhili Chen ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Detection ◽  
Detection Analysis ◽  
Analysis System

High-Throughput, High-Multiplex Digital Protein Detection with Attomolar Sensitivity

ACS Nano ◽  
2022 ◽  
Author(s):  
Connie Wu ◽  
Tyler J. Dougan ◽  
David R. Walt
Keyword(s):  
High Throughput ◽  
Protein Detection

High-Throughput Screening with HyperCyt® Flow Cytometry to Detect Small Molecule Formylpeptide Receptor Ligands

2005 ◽  
Vol 10 (4) ◽  
pp. 374-382 ◽  
Author(s):  
Susan M. Young ◽  
Cristian Bologa ◽  
Eric R. Prossnitz ◽  
Tudor I. Oprea ◽  
Larry A. Sklar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Receptor Ligands ◽  
Chemical Library ◽  
Assay Sensitivity ◽  
Compound Libraries ◽  
Analysis System ◽  
G Protein Coupled ◽  
Formylpeptide Receptor

High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt® is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein–coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (~2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [Ki]=14 μM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt® system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.

scholarly journals MspA Porin as a Local Nanopore Probe for Membrane-bound Proteins

2021 ◽  
Author(s):  
David P. Hoogerheide ◽  
Philip A. Gurnev ◽  
Jens Gundlach ◽  
Andrew Laszlo ◽  
Tatiana K. Rostovtseva ◽  
...  
Keyword(s):  
Protein Detection ◽  
Capture Process ◽  
Neuronal Protein ◽  
Intrinsically Disordered ◽  
Detection Analysis ◽  
Direct Discrimination ◽  
Crossover Behavior ◽  
Voltage Dependent ◽  
Conductance Changes ◽  
Nanopore Confinement

Nanopore sensing is based on detection and analysis of nanopore transient conductance changes induced by analyte capture. We have recently shown that α-Synuclein (αSyn), an intrinsically disordered, membrane-active, neuronal protein implicated in Parkinson disease, can be reversibly captured by the VDAC nanopore. The capture process is a highly voltage dependent complexation of the two proteins where transmembrane potential drives the polyanionic C-terminal domain of αSyn into VDAC--exactly the mechanism by which generic nanopore-based interrogation of proteins and polynucleotides proceeds. The complex formation, and the motion of αSyn in the nanopore, thus may be expected to be only indirectly dependent on the pore identity. Here, we confirm this prediction by demonstrating that when VDAC is replaced with a different transmembrane pore, the engineered mycobacterial porin M2MspA, all the qualitative features of the αSyn/nanopore interaction are preserved. The rate of αSyn capture by M2MspA rises exponentially with the applied field, while the residence time displays a crossover behavior, indicating that at voltages >50 mV M2MspA-bound αSyn largely undergoes translocation to the other side of the membrane. The translocation is directly confirmed using the selectivity tag method, in which the polyanionic C-terminal and neutral N-terminal regions of αSyn alter the selectivity of the M2MspA channel differently, allowing direct discrimination of translocation vs retraction for single αSyn molecules. We thus prove that the physical model of the motion of disordered protein chains in the nanopore confinement and the selectivity tag technique are not limited to VDAC but are broadly applicable to nanopore-based protein detection, analysis, and separation technologies.

scholarly journals Application of a Rapid and Integrated Analysis System (RIAS) as a High-Throughput Processing Tool for In Vitro ADME Samples by Liquid Chromatography/Tandem Mass Spectrometry

2011 ◽  
Vol 16 (3) ◽  
pp. 370-377 ◽  
Author(s):  
Andreas H. Luippold ◽  
Thomas Arnhold ◽  
Wolfgang Jörg ◽  
Beate Krüger ◽  
Roderich D. Süssmuth
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
High Throughput ◽  
Integrated Analysis ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Analysis System

Over the past decade, drug discovery programs have started to address the optimization of key ADME properties already at an early stage of the process. Hence, analytical chemists have been confronted with tremendously rising sample numbers and have had to develop methodologies accelerating quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS). This article focuses on the application of a generic and fully automated LC/MS/MS, named Rapid and Integrated Analysis System (RIAS), as a high-throughput platform for the rapid quantification of drug-like compounds in various in vitro ADME assays. Previous efforts were dedicated to the setup and feasibility study of a workflow-integrated platform combining a modified high-throughput liquid handling LC/MS/MS system controlled by a customized software interface and a customized data-processing and reporting tool. Herein the authors present an extension of this previously developed basic application to a broad set of ADME screening campaigns, covering CYP inhibition, Caco-2, and PAMPA assays. The platform is capable of switching automatically between various ADME assays, performs MS compound optimization if required, and provides a speed of 8 s from sample to sample, independently of the type of ADME assay. Quantification and peak review are adopted to the high-throughput environment and tested against a standard HPLC-ESI technology.

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