ArticleProceedings of the National Academy of Sciences of the United States of America2009
A site-specific, multiplexed kinase activity assay using stable-isotope dilution and high-resolution mass spectrometry.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 31 papers.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
31 citing papers in PubMed, 78 citations in OpenAlex.
- Maximizing Depth of PTM Coverage: Generating Robust MS Datasets for Computational Prediction Modeling.Methods in molecular biology (Clifton, N.J.) · 2022Review
- Article
- Drug Discovery in Liver Disease Using Kinome Profiling.International journal of molecular sciences · 2021Review
- An ultrasensitive fiveplex activity assay for cellular kinases.Scientific reports · 2019Article
- Analysis of the Human Kinome and Phosphatome by Mass Cytometry Reveals Overexpression-Induced Effects on Cancer-Related Signaling.Molecular cell · 2019Article
- Application of targeted mass spectrometry in bottom-up proteomics for systems biology research.Journal of proteomics · 2018Review
- Phosphoproteomics-Based Profiling of Kinase Activities in Cancer Cells.Methods in molecular biology (Clifton, N.J.) · 2018Article
- Strategy for Identification of Phosphorylation Levels of Low Abundance Proteins in Vivo for Which Antibodies Are not Available.Journal of cardiovascular development and disease · 2017Article
- Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.Molecular & cellular proteomics : MCP · 2017Article
- Global Phosphoproteomic Analysis of Insulin/Akt/mTORC1/S6K Signaling in Rat Hepatocytes.Journal of proteome research · 2017Article
- Multiplex Substrate Profiling by Mass Spectrometry for Kinases as a Method for Revealing Quantitative Substrate Motifs.Analytical chemistry · 2017Article
- Recent advances in methods to assess the activity of the kinome.F1000Research · 2017Review
- Sensing Enzymatic Activity by Exposure and Selection of DNA-Encoded Probes.Angewandte Chemie (International ed. in English) · 2016Article
- Recent findings and technological advances in phosphoproteomics for cells and tissues.Expert review of proteomics · 2015Review
- Systematic analysis of the phosphoproteome and kinase-substrate networks in the mouse testis.Molecular & cellular proteomics : MCP · 2014Article
- Structural basis for phosphorylation and lysine acetylation cross-talk in a kinase motif associated with myocardial ischemia and cardioprotection.The Journal of biological chemistry · 2014Article
- Stable isotope labeling of phosphoproteins for large-scale phosphorylation rate determination.Molecular & cellular proteomics : MCP · 2014Article
- NANOG is multiply phosphorylated and directly modified by ERK2 and CDK1 in vitro.Stem cell reports · 2014Article
- Protein analysis by shotgun/bottom-up proteomics.Chemical reviews · 2013Review
- Temporal profiling of lapatinib-suppressed phosphorylation signals in EGFR/HER2 pathways.Molecular & cellular proteomics : MCP · 2012Article
Corrections and comments
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
Abstract
Most kinases are capable of recognizing and phosphorylating peptides containing short, linear sequence motifs. To measure the activation state of many kinases from the same cell lysate, we created a multiplexed, mass-spectrometry-based in vitro kinase assay. Ninety chemically synthesized peptides derived from well-characterized peptide substrates and in vivo phosphorylation sites with either known or previously unidentified upstream kinases were reacted individually in a plate format with crude cell lysates and ATP. Phosphorylation rates were directly measured based on the addition of 90 same-sequence, site-specific phosphopeptides enriched in stable isotopes to act as ideal quantitative internal standards for analysis by liquid chromatography coupled to tandem mass spectrometry. This approach concurrently measured up to 90 site-specific peptide phosphorylation rates, reporting a diagnostic fingerprint for activated kinase pathways. We applied this unique kinome-activity profiling strategy in a variety of cellular settings, including mitogen stimulation, cell cycle, pharmacological inhibition of pathways, and to a panel of breast cancer cell lines. Finally, we identified the source of activity for a peptide (derived from a PI3K regulatory subunit) from our library. This peptide substrate demonstrated mitotic and tyrosine-specific phosphorylation, which was confirmed to be a novel Src family kinase site in vivo.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.