Online Magnetic Bead Dynamic Protein-Affinity Selection Coupled to LC−MS for the Screening of Pharmacologically Active Compounds

Abstract

The online, selective isolation of protein-ligand complexes using cobalt(II)-coated paramagnetic affinity beads (PABs) and subsequent liquid chromatography-mass spectrometry (LC-MS) determination of specifically bound ligands is described. After in-solution incubation of an analyte mixture with His-tagged target proteins, protein-analyte complexes are mixed with the Co(II)-PABs and subsequently injected into an in-house built magnetic trapping device. Bioactive ligands bound to the protein-Co(II)-PABs are retained in the magnetic field of the trapping device while inactive compounds are removed by washing with a pH 7.4 buffer. Active ligands are online eluted toward the LC-MS system using a pH shift. In the final step of the procedure, the protein-Co(II)-PABs are flushed to waste by temporarily lowering the magnetic field. The proof-of-principle is demonstrated by using commercially available Co(II)-PABs in combination with the His-tagged human estrogen-receptor ligand-binding domain. The system is characterized with a number of estrogenic ligands and nonbinding pharmaceutical compounds. The affinities of the test compounds varied from the high micromolar to the subnanomolar range. Typical detection limits are in the range from 20 to 80 nmol/L. The system is able to identify binders in mixtures of compounds, with an analysis time of 9.5 min per mixture. The standard deviation over 24 h is 9%.

Magnetic solids in analytical chemistry: A review

“…These methods have served to improve the detection limits of earlier techniques and have even been proposed as rapid screening methods [92,[145][146][147][148][149][150][151]. Tumorigenic polynuclear aromatic hydrocarbons such as ergosterol, sulfonamides or salicylic acid have been isolated by magnetic solid-phase extraction.…”

Online magnetic bead based dynamic protein affinity selection coupled to LC–MS for the screening of acetylcholine binding protein ligands

“…A 20 mg/mL magnetic bead suspension was prepared according to the recent paper of Jonker et al [4]. In brief, 120 L of bead suspension was washed three times with 400 L binding buffer and finally resuspended in 240 L of binding buffer resulting in a concentration of 20 mg/mL.…”

“…Several distinct MS approaches towards screening of bioactives have been developed allowing the evaluation of protein-ligand binding by MS [4]. (Pulsed) ultrafiltration technologies utilize a target protein in an ultrafiltration chamber [5].…”

“…The advantages here are that the incubation occurs under native target conditions in solution and that new target protein is used for every measurement. In recently published work, we utilized the last mentioned approach with immobilized metal affinity chromatographic (IMAC) paramagnetic beads (referred to as 'magnetic beads') as solid support material and the His-tagged estrogen receptor (ligand binding domain) as the target protein [4]. The methodology employs in vial and solution based formation of estrogen receptor-ligand complexes followed by addition of magnetic beads.…”

Fast identification of lipase inhibitors in oolong tea by using lipase functionalised Fe3O4 magnetic nanoparticles coupled with UPLC–MS/MS

“…Recently, magnetic nanoparticles (MNPs) have been found to be an excellent solid support for bio-macromolecules due to their unique ability to separate magnetically, low toxicity, high flexibility with chemically modifiable surface, large enzyme loading capacity, and good reusability. Ligand fishing based on MNPs has been applied to screen ligands from cellular extracts as well as from herbs (Jonker et al, 2009). Several types of natural compounds have been demonstrated to comply with the technique.…”