A magnetic bead-based protein kinase assay with dual detection techniques

Abstract

A novel magnetic bead-based protein kinase assay was developed using MALDI-TOF mass spectrometry (MALDI-TOF MS) and immuno-chemifluorescence as two independent detection techniques. Abltide substrate was immobilized onto magnetic beads via non-covalent biotin-streptavidin interactions. This non-covalent immobilization strategy facilitated peptide release and allowed MALDI-TOF MS analysis of substrate phosphorylation. The use of magnetic beads provided rapid sample handling and allowed secondary analysis by immuno-chemifluorescence to determine the degree of substrate phosphorylation. This dual detection technique was used to evaluate the inhibition of c-Abl kinase by imatinib and dasatinib. For each inhibitor, IC50 (half-maximal inhibitory concentration) values determined by these two different detection methods were consistent and close to values reported in the literature. The high-throughput potential of this new approach to kinase assays was preliminarily demonstrated by screening a chemical library consisting of 31 compounds against c-Abl kinase using a 96-well plate. In this proof-of-principle experiment, both MALDI-TOF MS and immuno-chemifluorescence were able to compare inhibitor potencies with consistent values. Dual detection may significantly enhance the reliability of chemical library screening and identify false positives and negatives. Formatted for 96-well plates and with high-throughput potential, this dual detection kinase assay may provide a rapid, reliable and inexpensive route to the discovery of small molecule drug leads.

A Magnetic Bead–Based Ligand Binding Assay to Facilitate Human Kynurenine 3-Monooxygenase Drug Discovery

“…In a drugscreening context, magnetic bead assays have been applied in a functional assay of protein kinase activity and in a highthroughput one-bead-one-compound screening system, among other assay systems. 9,10 Importantly, magnetic beads have been used for isolation of target proteins from crude bacterial and mammalian cell lysates, then incorporated in a specific assay system.…”

Photocleavable peptide–oligonucleotide conjugates for protein kinase assays by MALDI-TOF MS

“…We have previously demonstrated that magnetic beads are a powerful tool for hybrid and solid-phase protein kinase assays, offering separation and suitability to automated procedures as well as favorable analysis by MALDI-TOF MS. 24–25 To pull out protein kinase products from kinase reaction mixtures by magnetic beads through DNA hybridization for detecting degree of phosphorylation, we investigated the immobilization of the complementary thiol-modified oligonucleotide (5′-HS GTACTTCCTTAAACGACGCAGG-3′) on magnetic beads. First, to prevent nonspecific interactions, a poly(ethylene glycol) (PEG) spacer linker was grafted onto the surface of magnetic beads according as previously reported (Figure 4).…”

Photocleavable Peptide-Conjugated Magnetic Beads for Protein Kinase Assays by MALDI-TOF MS

“…The phosphorylation ratio of Abltide immobilized on magnetic beads was determined according to the previously described computational method (47). …”

“…As an alternative strategy to decrease background after phosphorylation, biotin-tagged peptide substrates can be captured onto streptavidin-coated magnetic beads (46) prior to MALDI-TOF analysis. We recently extended this work to create a practical kinase activity screening assay and demonstrated rapid determination of IC 50 for small-molecule kinase inhibitors (47). In this study, we have used Fmoc solid-phase peptide synthesis to install diblock linkers terminated by a photocleavable group onto paramagnetic particles, tethered substrate peptides, and validated these conjugates as reporters of protein kinase activity and inhibition using a MALDI-TOF readout (Figure 1).…”