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Issue 9, 2012
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Photocleavable peptide–oligonucleotide conjugates for protein kinase assays by MALDI-TOF MS

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Abstract

Robust methods for highly parallel, quantitative analysis of cellular protein tyrosine kinase activities may provide tools critically needed to decipher oncogenic signaling, discover new targeted drugs, diagnose cancer and monitor patients. Here, we describe proof-of-principle for a novel protein kinase assay with the potential to help overcome these challenges. MALDI-TOF mass spectrometry provides an ideal tool for label-free multiplexed analysis of peptide phosphorylation, but is poorly matched to homogeneous assays and complex samples. Thus, we conjugated a common oligonucleotide tag to multiple peptide substrates, offering efficient capture from solution-phase kinase reactions by annealing to the complementary sequence tethered to PEG-passivated superparamagnetic microparticles. To enable reversible conjugation, we developed a novel bifunctional cross-linker allowing simple and efficient preparation of photocleavable peptideoligonucleotide conjugates. After washing away contaminants and following photorelease, MALDI-TOF analysis yielded relative phosphorylation of each peptide with high sensitivity and specificity. Validating the hybridization-mediated multiplexed kinase assay, when three peptide substrate–oligonucleotide conjugates were mixed with the tyrosine kinase c-Abl and ATP, we readily observed their differential phosphorylation yet measured a common IC50 for the Abl kinase inhibitor imatinib. This new assay enables analysis of protein kinase activities in a multiplexed format amenable to screening inhibitors against multiple kinases in parallel, an important capability for drug discovery and predictive diagnostics.

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

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Supplementary files

Article information


Submitted
27 Apr 2012
Accepted
11 Jun 2012
First published
13 Jun 2012

Mol. BioSyst., 2012,8, 2395-2404
Article type
Paper

Photocleavable peptideoligonucleotide conjugates for protein kinase assays by MALDI-TOF MS

G. Zhou, F. Khan, Q. Dai, J. E. Sylvester and S. J. Kron, Mol. BioSyst., 2012, 8, 2395
DOI: 10.1039/C2MB25163A

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