Development of a Magnetic Microbead Affinity Selection Screen (MagMASS) Using Mass Spectrometry for Ligands to the Retinoid X Receptor-α

Abstract

To overcome limiting factors in mass spectrometry-based 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 non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well 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 extracts.

Ultra-large chemical libraries for the discovery of high-affinity peptide binders

“…Here, we show that magnetic bead capture-based AS-MS 33,34 is capable of identifying binders from libraries of up to~10 8 random synthetic peptides (Fig. 1b).…”

Synergy and antagonism in natural product extracts: when 1 + 1 does not equal 2

“…To improve the speed of screening, a Magnetic Microbead Affinity Selection Screening (MagMASS) protocol was developed, in which the protein target of interest is not free in solution, but rather is bound to magnetic beads. 175 To separate compounds with and without affinity for the given target, the receptor-bound fraction can be held in solution using a magnet. 175 In a recent study, PUF-MS and MagMASS were compared, and both screening methods were found to reliably identify ligands of a specific molecular target from complex botanical matrices.…”

“…175 To separate compounds with and without affinity for the given target, the receptor-bound fraction can be held in solution using a magnet. 175 In a recent study, PUF-MS and MagMASS were compared, and both screening methods were found to reliably identify ligands of a specific molecular target from complex botanical matrices. 175 MagMASS showed a 6-fold faster separation of bound and unbound compounds when compared to PUF-MS and is compatible with a 96-well plate format.…”

Label‐free Bio‐affinity Mass Spectrometry for Screening and Locating Bioactive Molecules

“…However, a previous report (Yasuda et al, 2011) showed covalent immobilization led to the destruction of the activity of enzymes. The team of van Breemen (Rush et al, 2017) made a comparison between a magnetic microbead affinity‐MS‐based screening method and the PUF‐MS method for screening of retinoid X receptors (RXRα) inhibitors from botanical extracts and combinatorial libraries. Protein consumption during magnetic microbead affinity‐MS screening is minimal, requiring only 100 pmol per well.…”