A magnetic bead-based protein kinase assay with dual detection techniques.
A novel magnetic bead-based protein kinase assay was developed using MALDI-TOF mass spectrometry (MALDI-TOF MS) and immunochemifluorescence as two independent detection techniques. Abltide substrate was immobilized onto magnetic beads via noncovalent biotin-streptavidin interactions. This noncovalent 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 immunochemifluorescence 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 immunochemifluorescence 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.
Zhou
Guangchang
G
Ludwig Center for Metastasis Research, The University of Chicago, IL 60637, USA.
Sylvester
Juliesta E
JE
Wu
Ding
D
Veach
Darren R
DR
Kron
Stephen J
SJ
D000906:Antibodies / Q000276:immunology; D001549:Benzamides; D000069439:Dasatinib; D000068877:Imatinib Mesylate; D007118:Immunoassay / Q000379:methods; D008163:Luminescent Measurements / Q000379:methods*; D008280:Magnetics; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010879:Piperazines / Q000737:chemistry; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D016315:Proto-Oncogene Proteins c-abl / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013844:Thiazoles / Q000737:chemistry
D000906:Antibodies; D001549:Benzamides; D010455:Peptides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; D000068877:Imatinib Mesylate; D016315:Proto-Oncogene Proteins c-abl; D000069439:Dasatinib
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