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

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

Content Provider	Royal Society of Chemistry (RSC)
Author	Zhou, Guangchang Khan, Faraz Kron, Stephen J. Sylvester, Juliesta E. Dai, Qing
Copyright Year	2012
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 peptide–oligonucleotide 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.
Starting Page	2395
Ending Page	2404
Page Count	10
File Format	HTM / HTML PDF
ISSN	1742206X
Volume Number	8
Issue Number	9
Journal	Molecular BioSystems
DOI	10.1039/c2mb25163a
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Multiplexing Phosphorylation Imatinib ABL (gene) Abl Superparamagnetism Protein kinase inhibitor Protein Tyrosine kinase Protein kinase Ionization Kinase IC50 Oligonucleotide Drug discovery ATP Mass spectrometry Peptide Adenosine triphosphate Cancer Nucleic acid thermodynamics
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Biotechnology

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