High-Throughput Mass Spectrometry for Hit Identification: Current Landscape and Future Perspectives

2021 ◽  
Vol 26 (2) ◽  
pp. 168-191
Author(s):  
David G. McLaren ◽  
Vinit Shah ◽  
Thomas Wisniewski ◽  
Lucien Ghislain ◽  
Chang Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
Direct Detection ◽  
Label Free ◽  
Physiological Relevance ◽  
Wide Range ◽  
And Performance ◽  
Sensitivity Specificity

For nearly two decades mass spectrometry has been used as a label-free, direct-detection method for both functional and affinity-based screening of a wide range of therapeutically relevant target classes. Here, we present an overview of several established and emerging mass spectrometry platforms and summarize the unique strengths and performance characteristics of each as they apply to high-throughput screening. Multiple examples from the recent literature are highlighted in order to illustrate the power of each individual technique, with special emphasis given to cases where the use of mass spectrometry was found to be differentiating when compared with other detection formats. Indeed, as many of these examples will demonstrate, the inherent strengths of mass spectrometry—sensitivity, specificity, wide dynamic range, and amenability to complex matrices—can be leveraged to enhance the discriminating power and physiological relevance of assays included in screening cascades. It is our hope that this review will serve as a useful guide to readers of all backgrounds and experience levels on the applicability and benefits of mass spectrometry in the search for hits, leads, and, ultimately, drugs.

scholarly journals The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond

2015 ◽  
Vol 21 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Carl Haslam ◽  
John Hellicar ◽  
Adrian Dunn ◽  
Arne Fuetterer ◽  
Neil Hardy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Direct Detection ◽  
Sample Volume ◽  
Model Systems ◽  
Analysis Time ◽  
Label Free ◽  
Maldi Tof

Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction–based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in <10 s. While dramatically faster than liquid chromatography–coupled MS, an analysis time of 8–10 s is still considered relatively slow for full-diversity high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection. However, sample preparation and deposition onto the MALDI target, as well as interference from matrix ions, have been considered limitations for the use of MALDI for screening assays. Here we describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, we have generated robust data in a 1536 format and also increased sample deposition to 6144 samples per target. Using these methods, we demonstrate that this technology can deliver fast sample analysis time with low sample volume, and data comparable to that of current RapidFire assays.

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

2017 ◽  
Vol 22 (10) ◽  
pp. 1246-1252 ◽  
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.

scholarly journals Identification of an Arginase II Inhibitor via RapidFire Mass Spectrometry Combined with Hydrophilic Interaction Chromatography

2018 ◽  
Vol 24 (4) ◽  
pp. 457-465 ◽  
Author(s):  
Wataru Asano ◽  
Yu Takahashi ◽  
Motoaki Kawano ◽  
Yoshiji Hantani
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Hydrophilic Interaction Chromatography ◽  
Label Free ◽  
Hydrophilic Interaction ◽  
High Concentration ◽  
Allosteric Site ◽  
Arginase Ii

Peripheral arterial disease (PAD) is an occlusive disease that can lead to atherosclerosis. The involvement of arginase II (Arg II) in PAD progression has been proposed. However, no promising drugs targeting Arg II have been developed to date for the treatment of PAD. In this study, we established a method for detecting the activity of Arg II via high-throughput label-free RapidFire mass spectrometry using hydrophilic interaction chromatography, which enables the direct measurement of l-ornithine produced by Arg II. This approach facilitated a robust high-concentration screening of fragment compounds and the identification of a fragment that inhibits the activity of Arg II. We further confirmed binding of the fragment to the potential allosteric site of Arg II using a surface plasmon resonance assay. We concluded that the identified fragment is a promising compound that may lead to novel drugs to treat PAD, and our method for detecting the activity of Arg II can be applied to large-scale high-throughput screening to identify other structural types of Arg II inhibitors.

scholarly journals Assay Development and Screening of Human DGAT1 Inhibitors with an LC/MS-Based Assay

2010 ◽  
Vol 15 (6) ◽  
pp. 695-702 ◽  
Author(s):  
Ji-Hu Zhang ◽  
Thomas P. Roddy ◽  
Pei-I Ho ◽  
Christopher R. Horvath ◽  
Chad Vickers ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Biological Response ◽  
Fluorescent Labeling ◽  
Assay Development ◽  
Label Free ◽  
Chemical Library ◽  
Label Free Detection

Many attractive targets for therapeutic intervention are enzymes that catalyze biological reactions involving small molecules such as lipids, fatty acids, amino acid derivatives, nucleic acid derivatives, and cofactors. Some of the reactions are difficult to detect by methods commonly used in high-throughput screening (HTS) without specific radioactive or fluorescent labeling of substrates. In addition, there are instances when labeling has a detrimental effect on the biological response. Generally, applicable assay methodologies for detection of such reactions are thus required. Mass spectrometry (MS), being a label-free detection tool, has been actively pursued for assay detection in HTS in the past several years. The authors have explored the use of multiparallel liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for high-throughput detection of biochemical reactions. In this report, we describe in detail the assay development and screening with a LC/MS-based system for inhibitors of human diacylglycerol acyltransferase (DGAT1) with a chemical library of approximately 800,000 compounds. Several strategies and process improvements have been investigated to overcome technical challenges such as data variation and throughput. Results indicated that, through these innovative approaches, the LC/MS-based screening method is both feasible and suitable for high-throughput primary screening.

scholarly journals Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hubert J. Sijben ◽  
Wieke M. van Oostveen ◽  
Peter B. R. Hartog ◽  
Laura Stucchi ◽  
Andrea Rossignoli ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Norepinephrine Transporter ◽  
Functional Assay ◽  
Substrate Uptake ◽  
Label Free ◽  
Screening Assay ◽  
Fluorescent Substrate ◽  
High Throughput Screening Assay ◽  
Wide Range

AbstractThe human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET—norepinephrine (NE), dopamine (DA) and epinephrine (EP)—were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z′ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

scholarly journals Label-Free High-Throughput Screening Assay for the Identification of Norepinephrine Transporter (NET / SLC6A2) Inhibitors

2021 ◽  
Author(s):  
Hubert Sijben ◽  
Wieke van Oostveen ◽  
Peter Hartog ◽  
Laura Stucchi ◽  
Giovanna Maresca ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Norepinephrine Transporter ◽  
Functional Assay ◽  
Substrate Uptake ◽  
Label Free ◽  
Screening Assay ◽  
Fluorescent Substrate ◽  
High Throughput Screening Assay ◽  
Wide Range

Abstract The human norepinephrine transporter (NET) is an established drug target for a wide range of neurological disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET inducibly expressed in a modified HEK293-JumpIn cell line. Three endogenous substrates of NET – norepinephrine (NE), dopamine (DA) and epinephrine (EP) – were each assessed in characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z’ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

scholarly journals High-Throughput Screening by Mass Spectrometry: Comparison with the Scintillation Proximity Assay with a Focused-File Screen of AKT1/PKBα

2007 ◽  
Vol 12 (4) ◽  
pp. 473-480 ◽  
Author(s):  
Andrea K. Quercia ◽  
William A. Lamarr ◽  
Jayhyuk Myung ◽  
Can C. Özbal ◽  
James A. Landro ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Label Free ◽  
False Negatives ◽  
Scintillation Proximity Assay ◽  
Therapy Target ◽  
Hit Identification ◽  
Selection Of

Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire™) with the scintillation proximity assay (SPA). The cancer therapy target AKT1/PKBα was screened against a focused library of kinase inhibitors and IC50 values determined for all compounds that exhibit > 50% inhibition. A selection of additional compounds that exhibited ≤ 50% inhibition in the primary screen was chosen as controls to confirm inactives. The selection of compounds is expected to identify common actives, common inactives, false positives, and false negatives. Agreement is found between HTMS and SPA in terms of primary hit identification and hit confirmation. ( Journal of Biomolecular Screening 2007:473-480)

scholarly journals Syntrophic co-culture amplification of production phenotype for high-throughput screening of microbial strain libraries

2019 ◽  
Author(s):  
Tatyana E. Saleski ◽  
Alissa R. Kerner ◽  
Meng Ting Chung ◽  
Corine M. Jackman ◽  
Azzaya Khasbaatar ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Parent Strain ◽  
Dynamic Range ◽  
Random Mutagenesis ◽  
Spatial Separation ◽  
List Type ◽  
Target Molecule ◽  
Wide Range ◽  
Target Molecules

AbstractMicrobes can be engineered to synthesize a wide array of bioproducts, yet production phenotype evaluation remains a frequent bottleneck in the design-build-test cycle where strain development requires iterative rounds of library construction and testing. Here, we presentSyntrophicCo-cultureAmplification ofProduction phenotype (SnoCAP). Through a metabolic cross-feeding circuit, the production level of a target molecule is translated into highly distinguishable co-culture growth characteristics, which amplifies differences in production into highly distinguishable growth phenotypes. We demonstrate SnoCAP with the screening ofEscherichia colistrains for production of two target molecules: 2-ketoisovalerate, a precursor of the drop-in biofuel isobutanol, and L-tryptophan. The dynamic range of the screening can be tuned by employing an inhibitory analog of the target molecule. Screening based on this framework requires compartmentalization of individual producers with the sensor strain. We explore three formats of implementation with increasing throughput capability: confinement in microtiter plates (102-104assays/experiment), spatial separation on agar plates (104-105assays/experiment), and encapsulation in microdroplets (105-107assays/experiment). Using SnoCAP, we identified an efficient isobutanol production strain from a random mutagenesis library, reaching a final titer that is 5-fold higher than that of the parent strain. The framework can also be extended to screening for secondary metabolite production using a push-pull strategy. We expect that SnoCAP can be readily adapted to the screening of various microbial species, to improve production of a wide range of target molecules.HighlightsA high-throughput screening platform based on cross-feeding auxotrophs was developed.Compartmentalization was implemented in three formats: microplates, agar plates, and microdroplets.Utility of the screening was demonstrated for two proof-of-concept target molecules: 2-ketoisovalerate and L-tryptophan.The assay dynamic range was tuned by addition of an inhibitory analog.The screening was applied to identify a strain from a chemically mutagenized library that produces 5-fold higher isobutanol titer than the parent strain.

Sign in / Sign up

Export Citation Format

Share Document