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Updated: May 29, 2026

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Detecting protein higher-order structural changes using kinase as a phospho-labeler
Asato Maeda1, Kosuke Ogata1, Yasushi Ishihama2
1Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
None:
We developed an approach to detect protein structural changes on a proteome-wide scale by phosphate labeling through in vitro kinase reactions. This structural proteomics approach combines residue-specific labeling based on kinase substrate recognition with quantitative phosphoproteomics using phosphopeptide enrichment, enabling site-resolved profiling of structural alterations. Using myoglobin with and without heat denaturation and HEK293T cell extracts with and without protease treatment, we demonstrated that phosphorylation efficiency reflects differences in substrate protein structure. Moreover, by comparing phosphorylation efficiencies before and after RNA digestion, we successfully identified proteome-wide structural changes in non-denatured cell extracts. This approach enables a residue-specific readout of structural dynamics within the intracellular proteome.
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