An efficient and label-free LC-MS/MS method for assessing drug’s activity at dopamine and serotonin transporters using transporter-transfected HEK293T cells

2021 ◽  
pp. 026988112110085
Author(s):  
Zebin Lin ◽  
Chenyang Liu ◽  
Enshan Fan ◽  
Yurong Zhang ◽  
Shuiqing Zheng ◽  
...  
Keyword(s):  
Attractive Alternative ◽  
Tandem Mass ◽  
Label Free ◽  
Psychoactive Substances ◽  
Human Embryonic Kidney ◽  
Uptake Inhibition ◽  
Drug Activity ◽  
Functional Assays ◽  
Assay Procedure ◽  
Radiolabeled Compounds

Background: Dopamine transporter (DAT) and serotonin transporter (SERT) are targets for many psychoactive substances. Functional assays including uptake inhibition and release assays often involve radiolabeled compounds like [3H]-dopamine and [3H]-serotonin to assess drug activity at transporters, which have high requirements on handling radioactive samples. Aims: The aim of this study was to establish a label-free method to assess drug activity at DAT and SERT. Methods: A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was established using transporter-transfected human embryonic kidney 293T (HEK293T) cells. This method was evaluated by testing the effects of amphetamine and cocaine in the assay procedure. Results: The limits of detection of this method were 0.2 nM for both dopamine (DA) and serotonin (5-HT), with good linearities in the range of 0.5–160 nM. Amphetamine and cocaine’s IC50 and EC50 on DAT and SERT determined by this method were consistent with previous reports. Conclusions: A rapid, reliable and label-free LC-MS/MS method for assessing drug activity was established, which affords an attractive alternative for those laboratories that do not have a radiation license or capabilities.

scholarly journals Determination of 19 Psychoactive Substances in Premortem and Postmortem Whole Blood Samples Using Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Separations ◽  
2021 ◽  
Vol 8 (6) ◽  
pp. 78
Author(s):  
Sevasti Karampela ◽  
Jessica Smith ◽  
Irene Panderi
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Formic Acid ◽  
Whole Blood ◽  
High Performance ◽  
Tandem Mass ◽  
Psychoactive Substances ◽  
Blood Samples ◽  
Whole Blood Samples

An ever-increasing need exists within the forensic laboratories to develop analytical processes for the qualitative and quantitative determination of a broad spectrum of new psychoactive substances. Phenylethylamine derivatives are among the major classes of psychoactive substances available on the global market and include both amphetamine analogues and synthetic cathinones. In this work, an ultra-high-performance liquid chromatography-positive ion electrospray ionization tandem mass spectrometric method (UHPLC-ESI-MS/MS) has been developed and fully validated for the determination of 19 psychoactive substances, including nine amphetamine-type stimulants and 10 synthetic cathinone derivatives, in premortem and postmortem whole blood. The assay was based on the use of 1 mL premortem or postmortem whole blood, following solid phase extraction prior to the analysis. The separation was achieved on a Poroshell 120 EC-C18 analytical column with a gradient mobile phase of 0.1% formic acid in acetonitrile and 0.1% formic acid in water in 9 min. The dynamic multiple reaction monitoring used in this work allowed for limit of detection (LOD) and lower limit of quantitation (LOQ) values of 0.5 and 2 ng mL−1, respectively, for all analytes both in premortem and postmortem whole blood samples. A quadratic calibration model was used for the 12 quantitative analytes over the concentration range of 20–2000 ng mL−1, and the method was shown to be precise and accurate both in premortem and postmortem whole blood. The method was applied to the analysis of real cases and proved to be a valuable tool in forensic and clinical toxicology.

scholarly journals Fast Proteome Identification and Quantification from Data-Dependent Acquisition–Tandem Mass Spectrometry (DDA MS/MS) Using Free Software Tools

2019 ◽  
Vol 2 (1) ◽  
pp. 8 ◽  
Author(s):  
Jesse Meyer
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Gene Knockout ◽  
Tandem Mass ◽  
Label Free ◽  
Software Packages ◽  
Complex Process ◽  
Data Files ◽  
Using Data ◽  
Free Quantification

The identification of nearly all proteins in a biological system using data-dependent acquisition (DDA) tandem mass spectrometry has become routine for organisms with relatively small genomes such as bacteria and yeast. Still, the quantification of the identified proteins may be a complex process and often requires multiple different software packages. In this protocol, I describe a flexible strategy for the identification and label-free quantification of proteins from bottom-up proteomics experiments. This method can be used to quantify all the detectable proteins in any DDA dataset collected with high-resolution precursor scans and may be used to quantify proteome remodeling in response to drug treatment or a gene knockout. Notably, the method is statistically rigorous, uses the latest and fastest freely-available software, and the entire protocol can be completed in a few hours with a small number of data files from the analysis of yeast.

scholarly journals A Label-Free LC/MS/MS-Based Enzymatic Activity Assay for the Detection of Genuine Caspase Inhibitors and SAR Development

2013 ◽  
Vol 18 (8) ◽  
pp. 868-878 ◽  
Author(s):  
Michel C. Maillard ◽  
Celia Dominguez ◽  
Mark J. Gemkow ◽  
Florian Krieger ◽  
Hyunsun Park ◽  
...  
Keyword(s):  
Published Data ◽  
Tandem Mass ◽  
Label Free ◽  
Activity Assay ◽  
Fluorogenic Substrates ◽  
Enzymatic Assays ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Enzymatic Activity Assay ◽  
Assay Interference

The resurgence of interest in caspases (Csp) as therapeutic targets for the treatment of neurodegenerative diseases prompted us to examine the suitability of published nonpeptidic Csp-3 and Csp-6 inhibitors for our medicinal chemistry programs. To support this effort, fluorescence-based Csp-2, Csp-3, and Csp-6 enzymatic assays were optimized for robustness against apparent enzyme inhibition caused by redox-cycling or aggregating compounds. The data obtained under these improved conditions challenge the validity of previously published data on Csp-3 and Csp-6 inhibitors for all but one series, namely, the isatins. Furthermore, in this series, it was observed that the nature of the rhodamine-labeled substrate, typically used to measure caspase activity, interfered with the pharmacological sensitivity of the Csp-2 assay. As a result, a liquid chromatography/tandem mass spectrometry–based assay that eliminates label-dependent assay interference was developed for Csp-2 and Csp-3. In these label-free assays, the activity values of the Csp-2 and Csp-3 reference inhibitors were in agreement with those obtained with the fluorogenic substrates. However, isatin 10a was 50-fold less potent in the label-free Csp-2 assay compared with the rhodamine-based fluorescence format, thus proving the need for an orthogonal readout to validate inhibitors in this class of targets highly susceptible to artifactual inhibition.

scholarly journals DirectMS1: MS/MS-free identification of 1000 proteins of cellular proteomes in 5 minutes

2019 ◽  
Author(s):  
Mark V. Ivanov ◽  
Julia A. Bubis ◽  
Vladimir Gorshkov ◽  
Irina A. Tarasova ◽  
Lev I. Levitsky ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Biomarker Discovery ◽  
Tandem Mass ◽  
Label Free ◽  
Hela Cell Line ◽  
Sequence Coverage ◽  
Proteome Coverage ◽  
Successful Attempt ◽  
Higher Sensitivity ◽  
Lengthy Analysis

AbstractProteome characterization relies heavily on tandem mass spectrometry (MS/MS) and is thus associated with instrumentation complexity, lengthy analysis time, and limited duty-cycle. It was always tempting to implement approaches which do not require MS/MS, yet, they were constantly failing in achieving meaningful depth of quantitative proteome coverage within short experimental times, which is particular important for clinical or biomarker discovery applications. Here, we report on the first successful attempt to develop a truly MS/MS-free and label-free method for bottom-up proteomics. We demonstrate identification of 1000 protein groups for a standard HeLa cell line digest using 5-minute LC gradients. The amount of loaded sample was varied in a range from 1 ng to 500 ng, and the method demonstrated 10-fold higher sensitivity compared with the standard MS/MS-based approach. Due to significantly higher sequence coverage obtained by the developed method, it outperforms all popular MS/MS-based label-free quantitation approaches.

scholarly journals Combining Label‐free and Flourometric Functional Assays with Biophysical Binding Assays in Drug Discovery: A Comparison of Positives from an HTS Campaign

2009 ◽  
Vol 23 (S1) ◽  
Author(s):  
Matthew Wayne Olson ◽  
Jukka Kervinen ◽  
Carsten Schubert ◽  
Jennifer Kirkpatrick ◽  
James Kranz ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Label Free ◽  
Binding Assays ◽  
Functional Assays
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