scholarly journals Development of a High-Throughput Affinity Mass Spectrometry (AMS) Platform Using Laser Diode Thermal Desorption Ionization Coupled to Mass Spectrometry (LDTD-MS)

2020 ◽  
pp. 247255522097959
Author(s):  
Aniruddha Sahasrabuddhe ◽  
Dylan Oakley ◽  
Kui Chen ◽  
John D. McCarter
Keyword(s):  
Mass Spectrometry ◽  
Small Molecule ◽  
Thermal Desorption ◽  
Laser Diode ◽  
High Throughput ◽  
Solid Phase ◽  
Atmospheric Pressure Chemical Ionization ◽  
Ionization Source ◽  
Robust Detection ◽  
Compound Libraries

Affinity selection mass spectrometry (MS) or, simply, affinity mass spectrometry (AMS) is a label-free technology that has been used to identify high-affinity ligands of target proteins of interest by screening against small-molecule compound libraries and identifying molecules that are enriched in the presence of the target protein. We have previously applied Agilent Technology’s (Santa Clara, CA) RapidFire solid-phase extraction (SPE)-based high-throughput MS technology to screen small-molecule libraries using AMS. However, SPE-based technologies rely on fluidics for desalting and separation prior to mass analysis with attendant high solvent consumption, relatively high sample volume requirements, risk of sample carryover, and frequent maintenance. To address these challenges, we have established an AMS platform using a laser diode thermal desorption–atmospheric pressure chemical ionization (LDTD-APCI) ionization source (Phytronix, Quebec, Canada) coupled with a SCIEX 5600+ TripleTOF MS (Framingham, MA). We also validated a data-independent acquisition (DIA) Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) method for the robust detection and analysis of small-molecule affinity hits. An informatics platform developed in-house has resulted in a streamlined data analysis workflow for high-throughput AMS screening campaigns and reduced data processing time without compromising data quality. Finally, 68,000 compounds were screened in a single plate and affinity selected hits were confirmed in an orthogonal enzyme activity assay.

scholarly journals Coupling Laser Diode Thermal Desorption with Acoustic Sample Deposition to Improve Throughput of Mass Spectrometry–Based Screening

2015 ◽  
Vol 21 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Zuzana Haarhoff ◽  
Andrew Wagner ◽  
Pierre Picard ◽  
Dieter M. Drexler ◽  
Tatyana Zvyaga ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Thermal Desorption ◽  
Laser Diode ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Atmospheric Pressure Chemical Ionization ◽  
Label Free ◽  
Metabolite Formation ◽  
Cyp Inhibition ◽  
Sample Deposition

The move toward label-free screening in drug discovery has increased the demand for mass spectrometry (MS)–based analysis. Here we investigated the approach of coupling acoustic sample deposition (ASD) with laser diode thermal desorption (LDTD)–tandem mass spectrometry (MS/MS). We assessed its use in a cytochrome P450 (CYP) inhibition assay, where a decrease in metabolite formation signifies CYP inhibition. Metabolite levels for 3 CYP isoforms were measured as CYP3A4-1′-OH-midazolam, CYP2D6-dextrorphan, and CYP2C9-4′-OH-diclofenac. After incubation, samples (100 nL) were acoustically deposited onto a stainless steel 384-LazWell plate, then desorbed by an infrared laser directly from the plate surface into the gas phase, ionized by atmospheric pressure chemical ionization (APCI), and analyzed by MS/MS. Using this method, we achieved a sample analysis speed of 2.14 s/well, with bioanalytical performance comparable to the current online solid-phase extraction (SPE)–based MS method. An even faster readout speed was achieved when postreaction sample multiplexing was applied, where three reaction samples, one for each CYP, were transferred into the same well of the LazWell plate. In summary, LDTD coupled with acoustic sample deposition and multiplexing significantly decreased analysis time to 0.7 s/sample, making this MS-based approach feasible to support high-throughput screening (HTS) assays.

High-throughput solid-phase permethylation of glycans prior to mass spectrometry

2008 ◽  
Vol 22 (5) ◽  
pp. 721-734 ◽  
Author(s):  
Pilsoo Kang ◽  
Yehia Mechref ◽  
Milos V. Novotny
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Solid Phase

scholarly journals High Throughput in Silico Identification of Novel Phytochemical Inhibitors for the Master Regulator of Inflammation (TNFα)

2021 ◽  
Author(s):  
Pratap Kumar Parida ◽  
Dipak Paul ◽  
Debamitra Chakravorty
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
In Silico ◽  
Small Molecule Inhibitors ◽  
Binding Free Energy ◽  
Natural Compounds ◽  
Free Energy Calculations ◽  
Compound Libraries ◽  
Relative Binding

<p><a>The over expression of Tumor necrosis factor-α (TNFα) has been implicated in a variety of disease and is classified as a therapeutic target for inflammatory diseases (Crohn disease, psoriasis, psoriatic arthritis, rheumatoid arthritis).Commercially available therapeutics are biologics which are associated with several risks and limitations. Small molecule inhibitors and natural compounds (saponins) were identified by researchers as lead molecules against TNFα, however, </a>they were often associated with high IC50 values which can lead to their failure in clinical trials. This warrants research related to identification of better small molecule inhibitors by screening of large compound libraries. Recent developments have demonstrated power of natural compounds as safe therapeutics, hence, in this work, we have identified TNFα phytochemical inhibitors using high throughput <i>in silico </i>screening approaches of 6000 phytochemicals followed by 200 ns molecular dynamics simulations and relative binding free energy calculations. The work yielded potent hits that bind to TNFα at its dimer interface. The mechanism targeted was inhibition of oligomerization of TNFα upon phytochemical binding to restrict its interaction with TNF-R1 receptor. MD simulation analysis resulted in identification of two phytochemicals that showed stable protein-ligand conformations over time. The two compounds were triterpenoids: Momordicilin and Nimbolin A with relative binding energy- calculated by MM/PBSA to be -190.5 kJ/Mol and -188.03 kJ/Mol respectively. Therefore, through this work it is being suggested that these phytochemicals can be used for further <i>in vitro</i> analysis to confirm their inhibitory action against TNFα or can be used as scaffolds to arrive at better drug candidates.</p>

scholarly journals Drug discovery from Nature: automated high-quality sample preparation

2000 ◽  
Vol 22 (5) ◽  
pp. 149-157 ◽  
Author(s):  
Ralf Thiericke
Keyword(s):  
Drug Discovery ◽  
Sample Preparation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Solid Phase ◽  
Structural Diversity ◽  
Synthetic Compound ◽  
Natural Origin ◽  
Screening Programs ◽  
Compound Libraries

Secondary metabolites from plants, animals and microorganisms have been proven to be an outstanding source for new and innovative drugs and show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds:: often generated in time consuming and for the most part manual processes. As quality and quantity of the provided samples play a pivotal role in the success of high-throughput screening programs this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of a modified Zymark RapidTrace®SPE workstation an easy-to-handle and effective fractionation method has been developed which allows the generation of highquality samples from natural origin, fulfilling the requirements of an integration into high-throughput screening programs.

Rapid Determination of Chloramphenicol in Honey by Laser Diode Thermal Desorption Using Atmospheric Pressure Chemical Ionization-Tandem Mass Spectrometry

2013 ◽  
Vol 96 (3) ◽  
pp. 676-679 ◽  
Author(s):  
Grégory Blachon ◽  
Pierre Picard ◽  
Patrice Tremblay ◽  
Sarah Demers ◽  
Réal Paquin ◽  
...  
Keyword(s):  
Thermal Desorption ◽  
Laser Diode ◽  
Atmospheric Pressure ◽  
Chemical Ionization ◽  
Atmospheric Pressure Chemical Ionization ◽  
Rapid Determination ◽  
Internal Standard ◽  
Honey Sample ◽  
Pressure Chemical Ionization ◽  
Pressure Chemical

Abstract A high-throughput, rapid, and reliable method based on laser diode thermal desorption (LDTD) and atmospheric pressure chemical ionization coupled to tandem MS (APCI-MS/MS) was used to identify and quantify chloramphenicol (CAP) residues in honey. Sample pretreatment consisted of a liquid–liquid extraction of diluted honey in water with ethyl acetate containing stearic acid. After extraction, a 2 μL aliquot of the organic phase was deposited into a 96-well plate prior to detection by LDTD-APCI-MS/MS. The total analysis time was less than 6 s compared to several minutes for traditional chromatographic methods used for CAP detection, since no chromatographic separation was necessary. The extraction and analysis were made with honey samples of different color grading ranging from extra white to dark amber. The method showed good linearity (R2 = 0.99995) within a concentration range of 0.1 to 500 ng/g for CAP with D5-CAP as an internal standard. The RSD varied between 8 and 24% over the calibration range (n = 4). The method LOD for CAP in honey was 0.19 ng/g. This work demonstrates that LDTD-APCI-MS/MS could be used for fast and effective quantification of CAP in honey samples.

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