Photocleavable peptide-oligonucleotide conjugates for protein kinase assays by MALDI-TOF MSElectronic supplementary information (ESI) available: Characteristic data of the photocleavable cross-linker and its intermediates; MALDI spectra and HPLC chromatographs of the other purified peptide-oligonucleotide conjugates; MALDI data for in vitro inhibition assay. See DOI: 10.1039/c2mb25163a

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 IC 50 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. A sulfo-SMCC-based photocleavable cross-linker was synthesized and further used to prepare photocleavable peptide-oligonucleotide bioconjugates, which can re-release peptides under UV irradiation and facilitate detection by MALDI-TOF MS with high sensitivity and specificity.