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Sensitive multiplexed analysis of kinase activities and activity-based kinase identification.

ABSTRACT: Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry-based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.

SUBMITTER: Kubota K

PROVIDER: S-EPMC3129615 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Sensitive multiplexed analysis of kinase activities and activity-based kinase identification.

Kubota Kazuishi K Anjum Rana R Yu Yonghao Y Kunz Ryan C RC Andersen Jannik N JN Kraus Manfred M Keilhack Heike H Nagashima Kumiko K Krauss Stefan S Paweletz Cloud C Hendrickson Ronald C RC Feldman Adam S AS Wu Chin-Lee CL Rush John J Villén Judit J Gygi Steven P SP

Nature biotechnology 20091004 10

Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry-based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase ...[more]

PMID: 19801977

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Project description:Dysregulated glycotransferase enzymes in cancer cells produce aberrant glycans--some of which can help facilitate metastases. Within a cell, individual glycotransferases promiscuously help to construct dozens of unique glycan structures, making it difficult to comprehensively track their activity in biospecimens--especially where they are absent or inactive. Here, we describe an approach to deconstruct glycans in whole biospecimens then analytically pool together resulting monosaccharide-and-linkage-specific degradation products ("glycan nodes") that directly represent the activities of specific glycotransferases. To implement this concept, a reproducible, relative quantitation-based glycan methylation analysis methodology was developed that simultaneously captures information from N-, O-, and lipid linked glycans and is compatible with whole biofluids and homogenized tissues; in total, over 30 different glycan nodes are detectable per gas chromatography-mass spectrometry (GC-MS) run. Numerous nonliver organ cancers are known to induce the production of abnormally glycosylated serum proteins. Thus, following analytical validation, in blood plasma, the technique was applied to a group of 59 lung cancer patient plasma samples and age/gender/smoking-status-matched non-neoplastic controls from the Lung Cancer in Central and Eastern Europe (CEE) study to gauge the clinical utility of the approach toward the detection of lung cancer. Ten smoking-independent glycan node ratios were found that detect lung cancer with individual receiver operating characteristic (ROC) c-statistics ranging from 0.76 to 0.88. Two glycan nodes provided novel evidence for altered ST6Gal-I and GnT-IV glycotransferase activities in lung cancer patients. In summary, a conceptually novel approach to the analysis of glycans in unfractionated human biospecimens has been developed that, upon clinical validation for specific applications, may provide diagnostic and/or predictive information in glycan-altering diseases.

Dataset Information

Sensitive multiplexed analysis of kinase activities and activity-based kinase identification.

Publications

Sensitive multiplexed analysis of kinase activities and activity-based kinase identification.

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