High-throughput microscope for label-free detection of protein and small-molecule chemical microarrays

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

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MALDIViz: A Comprehensive Informatics Tool for MALDI-MS Data Visualization and Analysis

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555217727517 ◽

2017 ◽

Vol 22 (10) ◽

pp. 1246-1252 ◽

Cited By ~ 1

Author(s):

Kishore Kumar Jagadeesan ◽

Simon Ekström

Keyword(s):

Mass Spectrometry ◽

High Throughput ◽

High Throughput Screening ◽

Web Application ◽

Low Cost ◽

Maldi Ms ◽

Ionization Mass ◽

Label Free ◽

Label Free Detection ◽

R Shiny

Recently, mass spectrometry (MS) has emerged as an important tool for high-throughput screening (HTS) providing a direct and label-free detection method, complementing traditional fluorescent and colorimetric methodologies. Among the various MS techniques used for HTS, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides many of the characteristics required for high-throughput analyses, such as low cost, speed, and automation. However, visualization and analysis of the large datasets generated by HTS MALDI-MS can pose significant challenges, especially for multiparametric experiments. The datasets can be generated fast, and the complexity of the experimental data (e.g., screening many different sorbent phases, the sorbent mass, and the load, wash, and elution conditions) makes manual data analysis difficult. To address these challenges, a comprehensive informatics tool called MALDIViz was developed. This tool is an R-Shiny-based web application, accessible independently of the operating system and without the need to install any program locally. It has been designed to facilitate easy analysis and visualization of MALDI-MS datasets, comparison of multiplex experiments, and export of the analysis results to high-quality images.

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A Simple Displacement Aptamer Assay on Resistive Pulse Sensor for Small Molecule Detection

10.26434/chemrxiv.12855338.v1 ◽

2020 ◽

Author(s):

Rhushabh Maugi ◽

bernadette gamble ◽

david bunka ◽

Mark Platt

Keyword(s):

Small Molecules ◽

Small Molecule ◽

Label Free ◽

Resistive Pulse ◽

Pulse Sensor ◽

Label Free Detection ◽

Ssdna Aptamer ◽

Aptamer Assay ◽

Small Molecule Detection ◽

Surface Changes

A universal aptamer-based sensing strategy is proposed using DNA modified nanocarriers and Resistive Pulse Sensing for the rapid and label free detection of small molecules. The surface of a magnetic nanocarrier was first modified with a ssDNA aka linker which is designed to be partially complimentary in sequence to a ssDNA aptamer. The aptamer and linker form a stable dsDNA complex on the nanocarriers surface. Upon the addition of the target molecule, a conformational change takes place where the aptamer preferentially binds to the target over the linker; causing the aptamer to be released into solution. The RPS measures the change in velocity of the nanocarrier as its surface changes from dsDNA to ssDNA, and its velocity is used as a proxy for the concentration of the target. We illustrate the versatility of the assay by demonstrating the detection of the antibiotic Moxifloxacin, and chemotherapeutics Imatinib and Irinotecan.

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Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

Nucleic Acids Research ◽

10.1093/nar/gkaa849 ◽

2020 ◽

Vol 48 (20) ◽

pp. e120-e120 ◽

Cited By ~ 1

Author(s):

Obtin Alkhamis ◽

Weijuan Yang ◽

Rifat Farhana ◽

Haixiang Yu ◽

Yi Xiao

Keyword(s):

Ligand Binding ◽

Small Molecule ◽

High Throughput ◽

Dna Aptamer ◽

Label Free ◽

General Applicability ◽

Gold Standard Method ◽

Characterization Methods ◽

Increased Risk

Abstract In vitro aptamer isolation methods can yield hundreds of potential candidates, but selecting the optimal aptamer for a given application is challenging and laborious. Existing aptamer characterization methods either entail low-throughput analysis with sophisticated instrumentation, or offer the potential for higher throughput at the cost of providing a relatively increased risk of false-positive or -negative results. Here, we describe a novel method for accurately and sensitively evaluating the binding between DNA aptamers and small-molecule ligands in a high-throughput format without any aptamer engineering or labeling requirements. This approach is based on our new finding that ligand binding inhibits aptamer digestion by T5 exonuclease, where the extent of this inhibition correlates closely with the strength of aptamer-ligand binding. Our assay enables accurate and efficient screening of the ligand-binding profiles of individual aptamers, as well as the identification of the best target binders from a batch of aptamer candidates, independent of the ligands in question or the aptamer sequence and structure. We demonstrate the general applicability of this assay with a total of 106 aptamer-ligand pairs and validate these results with a gold-standard method. We expect that our assay can be readily expanded to characterize small-molecule-binding aptamers in an automated, high-throughput fashion.

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