Fluorescence Method for the Detection of Protein Kinase Activity by Using a Zirconium-Based Metal-Organic Framework as an Affinity Probe.

Abstract

In cell-signaling pathways, protein kinases are critical and ubiquitous regulators. Abnormal kinase activity leads to many major diseases; therefore, simple and efficient methods for detecting protein kinases are in high demand. This study proposed a simple, rapid fluorescence-based sensor for protein kinase activity analysis, using the zirconium-based metal organic framework UiO-66 as a highly efficient affinity probe. UiO-66 has a large specific surface area, good stability, and a large number of Zr defect sites, which can efficiently identify phosphorylation sites. UiO-66 is an ideal nanoreactor that can efficiently enrich phosphorylated peptides. Under optimal experimental conditions, the increased fluorescence intensity was directly proportional to the protein kinase activity. The lower limit of detection was 0.00005 U·μL-1. The assay could also be used for the screening of protein kinase inhibitors, could determine the activity of other kinds of kinases, and was universally applicable. This method was used for protein kinase activity detection in drug-stimulated MCF-7 cell lysates and demonstrated its potential applicability in kinase-related research.