Photocleavable peptide-conjugated magnetic beads for protein kinase assays by MALDI-TOF MS.
Peptides were immobilized onto superparamagnetic beads via photocleavable linkers. This enabled simple, rapid, and label-free protein kinase assays via MALDI-TOF MS detection of substrate peptide phosphorylation. Abltide, a model substrate for the Abl protein tyrosine kinase model, was coupled onto amine-terminated beads, incubated with ATP and recombinant c-Abl kinase, and released and further detected to determine phosphorylation. Abltide phosphorylation was found to depend significantly on the length and composition of linkers to the bead surface. Inserting a diblock spacer of poly(glycine) and poly(ethylene glycol) segments markedly enhanced phosphorylation. To validate the assay, the activity of two small-molecule kinase inhibitors, imatinib and dasatinib, which target the oncogenic mutant tyrosine kinase Bcr-Abl to treat chronic myeloid leukemia (CML), was tested. Examining inhibition of the purified c-Abl or Bcr-Abl in K562 CML cell extracts, IC(50) values were determined to be consistent with the literature. This simple, label-free, MALDI-based protein kinase assay can be readily adapted to allow multiplexed assays of multiple peptide substrates and/or analysis of alternative post-translational modifications as a tool for drug discovery and clinical testing.
Zhou
Guangchang
G
Ludwig Center for Metastasis Research, The University of Chicago, Chicago, Illinois 60637, USA.
Yan
Xiaoliang
X
Wu
Ding
D
Kron
Stephen J
SJ
D000595:Amino Acid Sequence; D057075:Enzyme Assays / Q000379:methods*; D005449:Fluorenes / Q000737:chemistry; D006801:Humans; D020014:K562 Cells; D008280:Magnetics; D008863:Microspheres; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D055668:Photochemical Processes; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
C496789:9-fluorenylmethoxycarbonyl; D005449:Fluorenes; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases