Department of Applied Chemistry Faculty of Engineering, Kyushu University, 744 Motooka Nishi, Fukuoka, 819-0395, Japan.
Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi, Fukuoka, 819-0395, Japan.
Anal Sci. 2021 Oct 10;37(10):1361-1366. doi: 10.2116/analsci.20P443. Epub 2021 Mar 12.
We have developed a novel FRET-based assay to monitor protein kinase activity using quantum dots (QDs) and fluorophore-labeled substrate peptides. To develop a FRET-based protein kinase assay, it is important to consider the phosphate group recognition strategy and to ensure that the FRET pairs are close enough because the FRET efficiency is highly dependent on the distance between the FRET pairs. Here, we incorporated a phos-tag, which captures phosphate groups strongly and selectively, into a protein kinase assay to recognize phosphorylation. Our detection system was composed of phos-tag-modified QDs and Cy5-labeled substrate peptides. Because the phos-tags capture phosphate groups by forming dinuclear complexes, the Cy5-labeled substrate peptides are captured by the phos-tags on the QD surface upon protein kinase-mediated phosphorylation, which induces FRET from the QDs to Cy5 because of the approximation of Cy5 to the QDs. On the basis of the difference of this FRET efficiency, we successfully measured protein kinase A activity, which demonstrated the feasibility of our assay.
我们开发了一种使用量子点（QDs）和荧光标记底物肽的新型 FRET 基蛋白激酶活性检测方法。为了开发基于 FRET 的蛋白激酶检测方法，重要的是要考虑磷酸基团识别策略，并确保 FRET 对足够接近，因为 FRET 效率高度依赖于 FRET 对之间的距离。在这里，我们将能够强烈且选择性地捕获磷酸基团的 phos-tag 整合到蛋白激酶检测中以识别磷酸化。我们的检测系统由 phos-tag 修饰的 QDs 和 Cy5 标记的底物肽组成。由于 phos-tags 通过形成双核配合物来捕获磷酸基团，因此在蛋白激酶介导的磷酸化后，Cy5 标记的底物肽被 QD 表面上的 phos-tags 捕获，这由于 Cy5 接近 QD 而引起从 QD 到 Cy5 的 FRET。基于这种 FRET 效率的差异，我们成功地测量了蛋白激酶 A 的活性，证明了我们的检测方法的可行性。
