ArticleNature protocols2009
Global identification of protein kinase substrates by protein microarray analysis.
Article in Nature protocols, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed.
- Proteomic approaches for protein kinase substrate identification in Apicomplexa.Molecular and biochemical parasitology · 2024Review
- CMGC Kinases in Health and Cancer.Cancers · 2023Review
- The protein methylation network in yeast: A landmark in completeness for a eukaryotic post-translational modification.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
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- Substrates of the MAPK Slt2: Shaping Yeast Cell Integrity.Journal of fungi (Basel, Switzerland) · 2022Review
- Article
- Mapping proteome-wide targets of protein kinases in plant stress responses.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- Methods and approaches to disease mechanisms using systems kinomics.Synthetic and systems biotechnology · 2018Review
- METTL21B Is a Novel Human Lysine Methyltransferase of Translation Elongation Factor 1A: Discovery by CRISPR/Cas9 Knockout.Molecular & cellular proteomics : MCP · 2017Article
- Quantifying Kinase-Specific Phosphorylation Stoichiometry Using Stable Isotope Labeling In a Reverse In-Gel Kinase Assay.Analytical chemistry · 2016Article
- AMP-activated Protein Kinase Up-regulates Mitogen-activated Protein (MAP) Kinase-interacting Serine/Threonine Kinase 1a-dependent Phosphorylation of Eukaryotic Translation Initiation Factor 4E.The Journal of biological chemistry · 2016Article
- Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human.Molecular & cellular proteomics : MCP · 2016Article
- The interactome of the atypical phosphatase Rtr1 in Saccharomyces cerevisiae.Molecular bioSystems · 2014Article
- Toward a systems-level view of dynamic phosphorylation networks.Frontiers in genetics · 2014Review
- Identification of direct tyrosine kinase substrates based on protein kinase assay-linked phosphoproteomics.Molecular & cellular proteomics : MCP · 2013Article
- Enhanced methylarginine characterization by post-translational modification-specific targeted data acquisition and electron-transfer dissociation mass spectrometry.Journal of the American Society for Mass Spectrometry · 2012Article
- Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.Proceedings of the National Academy of Sciences of the United States of America · 2012Article
- FLEXIQinase, a mass spectrometry-based assay, to unveil multikinase mechanisms.Nature methods · 2012Article
- A proteomic approach for the identification of novel lysine methyltransferase substrates.Epigenetics & chromatin · 2011Article
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Authors and funding
3 authors.
Funding
Abstract
Herein, we describe a protocol for the global identification of in vitro substrates targeted by protein kinases using protein microarray technology. Large numbers of fusion proteins tagged at their carboxy-termini are purified in 96-well format and spotted in duplicate onto amino-silane-coated slides in a spatially addressable manner. These arrays are incubated in the presence of purified kinase and radiolabeled ATP, and then washed, dried and analyzed by autoradiography. The extent of phosphorylation of each spot is quantified and normalized, and proteins that are reproducibly phosphorylated in the presence of the active kinase relative to control slides are scored as positive substrates. This approach enables the rapid determination of kinase-substrate relationship on a proteome-wide scale, and although developed using yeast, has since been adapted to higher eukaryotic systems. Expression, purification and printing of the yeast proteome require about 3 weeks. Afterwards, each kinase assay takes approximately 3 h to perform.
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