Wang Li-Juan, Yang Yong, Zhang Chun-Yang
Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China.
Anal Chem. 2015;87(9):4696-703. doi: 10.1021/ac504358q. Epub 2015 Apr 15.
Protein kinases play crucial roles in intracellular signal transduction and metabolic pathways, and the monitoring of protein kinase activity is essential to the understanding of fundamental biochemical processes and the clinical diagnosis. Here, we demonstrate the phosphorylation-directed assembly of a single quantum dot (QD)-based nanosensor for sensitive detection of cAMP-dependent protein kinase (PKA). This assay involves (1) the PKA-directed simultaneous phosphorylation and biotinylation of cyanine 5 (Cy5)-labeled substrate peptides, (2) the assembly of phosphorylated and biotinylated peptides onto the surface of the QD, and (3) the illumination of Cy5 by means of fluorescence resonance energy transfer (FRET) between the QD and Cy5. With an adenosine triphosphate (ATP) analogue, γ-biotin-ATP, as the phosphoryl donor, the PKA-catalyzed phosphorylation reaction incorporates the biotin-conjugated phosphate group into the substrate peptides to form the biotinylated peptides. The biotin entity subsequently drives the assembly of peptides onto the surface of streptavidin-functionalized QD to form the sandwiched Cy5-peptide-QD nanostructure, enabling the occurrence of FRET between the QD and Cy5. The FRET signal can be easily recorded by either the conventional fluorescence spectrometer or the total internal reflection fluorescence (TIRF) microscope. In contrast, the absence of PKA cannot lead to the formation of Cy5-peptide-QD complex and no Cy5 signal can be detected. This protein kinase-actuated FRET assay is straightforward, without the involvement of either washing or separation steps, and has a significant advantage of high sensitivity with a detection limit of 9.3 × 10(-6) U/μL. Moreover, this method can be used to estimate the half-maximal inhibitory concentration (IC50) value of PKA inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) and to monitor forskolin (Fsk)/3-isobutyl-1-methylxanthine (IBMX)-triggered activation of PKA in cell lysates, thus holding great potential for further applications in protein kinase-related biological researches and drug discovery.
蛋白激酶在细胞内信号转导和代谢途径中发挥着关键作用，对蛋白激酶活性的监测对于理解基本生化过程和临床诊断至关重要。在此，我们展示了一种基于单个量子点（QD）的纳米传感器的磷酸化导向组装，用于灵敏检测环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）。该检测方法包括：（1）PKA导向的对花菁5（Cy5）标记的底物肽进行同时磷酸化和生物素化；（2）将磷酸化和生物素化的肽组装到量子点表面；（3）通过量子点与Cy5之间的荧光共振能量转移（FRET）对Cy5进行光照。以三磷酸腺苷（ATP）类似物γ-生物素-ATP作为磷酰基供体，PKA催化的磷酸化反应将生物素共轭的磷酸基团掺入底物肽中，形成生物素化的肽。生物素实体随后驱动肽组装到链霉亲和素功能化的量子点表面，形成夹心式Cy5-肽-量子点纳米结构，使量子点与Cy5之间发生FRET。FRET信号可以很容易地通过传统荧光光谱仪或全内反射荧光（TIRF）显微镜记录。相比之下，没有PKA则无法导致Cy5-肽-量子点复合物的形成，并且检测不到Cy5信号。这种蛋白激酶驱动的FRET检测方法简单直接，无需洗涤或分离步骤，具有显著的高灵敏度优势，检测限为9.3×10⁻⁶ U/μL。此外，该方法可用于估计PKA抑制剂H-89（N-[2-(对溴肉桂氨基)乙基]-5-异喹啉磺酰胺二盐酸盐）的半数最大抑制浓度（IC50）值，并监测福斯可林（Fsk）/3-异丁基-1-甲基黄嘌呤（IBMX）触发的细胞裂解物中PKA的激活，因此在蛋白激酶相关生物学研究和药物发现的进一步应用中具有巨大潜力。
