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Title of Journal: J Am Soc Mass Spectrom

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Abbravation: Journal of The American Society for Mass Spectrometry

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Springer US

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DOI

10.1007/bf00540801

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1879-1123

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Development of a Magnetic Microbead Affinity Selec

Authors: Michael D Rush Elisabeth M Walker Gerd Prehna Tristesse Burton Richard B van Breemen
Publish Date: 2016/12/13
Volume: 28, Issue: 3, Pages: 479-485
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Abstract

To overcome limiting factors in mass spectrometrybased screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts magnetic microbead affinity selection screening MagMASS was developed The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries incubation with mixtures of molecules containing possible ligands a washing step that removes nonbound compounds while a magnetic field retains the beads in the microtiter well and an organic solvent release step followed by LCMS analysis Using retinoid X receptorα RXRα as an example which is a nuclear receptor and target for antiinflammation therapy as well as cancer treatment and prevention a MagMASS assay was developed and compared with an existing screening assay pulsed ultrafiltration PUFMS Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries The fulllength RXRα construct immobilized with amylose beads provided optimum results Additional enhancements of MagMASS were the application of 96well plates to enable automation use of UHPLC instead of HPLC for faster MS analyses and application of metabolomics software for faster automated data analysis Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extractsApproximately 50 of the cancer drugs used in the last 50 years have been inspired by natural products 1 The main sources of discovery of these therapeutic natural product compounds have been ethnomedicine and biological screening 2 3 While these sources have historically been valuable for drug discovery modern methods using reverse pharmacology drug discovery techniques also known as targetbased drug discovery have been underutilizing natural productsInstead of the slow process of testing for changes in a living cell or organism in response to a compound knowledge of the diseaserelevant receptorligand interactions allows for interrogation of a specific pathway For example a recombinant protein can be exposed to a single compound or mixture of compounds and any receptorligand interactions can be detected by using fluorescence enzymatic product formation thermal stability change or another method After a hit is detected the individual compound must be identified isolated and retested for activity and eventually developed as a drug lead However mixtures of natural products such as botanical extracts are rarely used in highthroughput screening primarily because of the extra expertise and time required to identify active constituents 3As with any reverse pharmacology drug discovery screen biological relevance depends upon the choice of target In this investigation the retinoid X receptorα RXRα was used as the target protein because it is an important nuclear receptor in the cancer protein network is a known target for multiple chemotherapy agents and unlike most other nuclear receptors few ligands are known 4 5 The endogenous ligand for RXRα is 9cis retinoic acid a derivative of vitamin A 6 A knockout mouse of RXRα develops a phenotype similar to vitamin A deficient mice with characteristic developmental morphology differentiation and cellular growth 7 One major mechanism of cell death linking RXRα activity with cancer is through its inhibition of NFE2 P45related factor 2 Nrf2 8 Treatment with an RXRα ligand can modulate the genes regulated by Nrf2 including critical cytoprotective genes implicated in cancer Several RXRα ligands such as bexarotene have received FDA approval to treat lymphoma 4 9 but these compounds have serious toxic side effects Therefore less toxic RXRα ligands are needed for therapeutic useRXRα has several structural requirements for biological activity and use in highthroughput screening After binding a ligand RXRα monomers change conformation and dimerize to form an active confirmation that can bind to DNA 10 11 Because RXRα dimerizes with onethird of all nuclear receptors including itself the challenge in targeting RXRα is obtaining specificity 12 13 The ligand binding domain LBD of RXRα is relatively independent both structurally and functionally and has been used instead of the fulllength construct to study many structural and functional aspects of RXRα 14 15 Both fulllength RXRα and its LBD were considered in this paperComparison of PUFMS and MagMASS During affinity selection screening ligands yellow triangles but not other low molecular weight compounds purple stars bind to a macromolecular target RXRα blue a In PUFMS the ligand–receptor complexes are separated in solution from nonbinding compounds by filtration through an ultrafiltration membrane grey dashed line Ligands are released by denaturing the receptor and recovered in the ultrafiltrate for LCMS analysis b During MagMASS an external magnetic force is applied to secure the magnetic beads orange ovals containing immobilized receptor and affinity bound ligand while the unbound compounds are washed away Ligands are released using organic solvent and/or a pH change and separated from the magnetic beads for UHPLCMS analysis


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Other Papers In This Journal:

  1. Distonic Ions: Editorial
  2. On the Efficiency of NHS Ester Cross-Linkers for Stabilizing Integral Membrane Protein Complexes
  3. Dynamic Interchanging Native States of Lymphotactin Examined by SNAPP-MS
  4. Quantitative Assessment of Protein Structural Models by Comparison of H/D Exchange MS Data with Exchange Behavior Accurately Predicted by DXCOREX
  5. Reflections on Charge State Distributions, Protein Structure, and the Mystical Mechanism of Electrospray Ionization
  6. CYCLONE—A Utility for De Novo Sequencing of Microbial Cyclic Peptides
  7. Mass Spectrometry-Based Quantification of Pseudouridine in RNA
  8. Statistical Examination of the a and a + 1 Fragment Ions from 193 nm Ultraviolet Photodissociation Reveals Local Hydrogen Bonding Interactions
  9. Perspective on Electrospray Ionization and Its Relation to Electrochemistry
  10. Untargeted Metabolomics Strategies—Challenges and Emerging Directions
  11. Structural Investigation of Protonated Azidothymidine and Protonated Dimer
  12. Application of Probe Electrospray Ionization Mass Spectrometry (PESI-MS) to Clinical Diagnosis: Solvent Effect on Lipid Analysis
  13. Ion-Molecule Clustering in Differential Mobility Spectrometry: Lessons Learned from Tetraalkylammonium Cations and their Isomers
  14. Charge Detection Mass Spectrometry for Single Ions with an Uncertainty in the Charge Measurement of 0.65 e
  15. Super-Atmospheric Pressure Electrospray Ion Source: Applied to Aqueous Solution
  16. Probing the Electron Capture Dissociation Mass Spectrometry of Phosphopeptides with Traveling Wave Ion Mobility Spectrometry and Molecular Dynamics Simulations
  17. Efficient Covalent Bond Formation in Gas-Phase Peptide–Peptide Ion Complexes with the Photoleucine Stapler
  18. Ion Trap Electric Field Characterization Using Slab Coupled Optical Fiber Sensors
  19. Picoelectrospray Ionization Mass Spectrometry Using Narrow-Bore Chemically Etched Emitters
  20. The H-Index of ‘An Approach to Correlate Tandem Mass Spectral Data of Peptides with Amino Acid Sequences in a Protein Database’
  21. Predicting Compensation Voltage for Singly-charged Ions in High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS)
  22. Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations
  23. Characterization of Tyrosine Nitration and Cysteine Nitrosylation Modifications by Metastable Atom-Activation Dissociation Mass Spectrometry
  24. Deconstructing Desorption Electrospray Ionization: Independent Optimization of Desorption and Ionization by Spray Desorption Collection
  25. Matrix Assisted Ionization in Vacuum, a Sensitive and Widely Applicable Ionization Method for Mass Spectrometry
  26. Localization of Post-Translational Modifications in Peptide Mixtures via High-Resolution Differential Ion Mobility Separations Followed by Electron Transfer Dissociation
  27. MALDI Mass Spectrometric Imaging of Lipids in Rat Brain Injury Models
  28. High Production of Small Organic Dicarboxylate Dianions by DESI and ESI
  29. Automated Lipid A Structure Assignment from Hierarchical Tandem Mass Spectrometry Data
  30. Automated Lipid A Structure Assignment from Hierarchical Tandem Mass Spectrometry Data
  31. Transitioning from Targeted to Comprehensive Mass Spectrometry Using Genetic Algorithms

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