Anal Chem. 2011 Dec 15;83(24):9230-3. doi: 10.1021/ac202723h. Epub 2011 Nov 18.
Mutations of the small GTP-binding protein Ras have been commonly found in tumors, and Ras oncogenes have been established to be involved in the early steps of cancerogenesis. The detection of Ras activity is critical in the determination of the cell signaling events controlling cell growth and differentiation. Therefore, development of improved methods for primary screening of novel potential drugs that target small GTPase or their regulators and their signaling pathways is important. Several assays have been developed for small GTPases studies, but all these methods have limitations for a high-throughput screening (HTS) use. Multiple steps including separation, use of radioactive labels or time-consuming immunoblotting, and a need of large quantities of purified proteins are decreasing the user-friendliness of these methods. Here, we have developed a homogeneous H-Ras activity assay based on a single-label utilizing the homogeneous quenching resonance energy transfer technique (QRET). In the QRET method, the binding of a terbium-labeled GTP (Tb-GTP) to small GTPase protein H-Ras protects the signal of the label from quenching, whereas the signal of the nonbound fraction of Tb-GTP is quenched by a soluble quencher. This enables a rapid determination of the changes in the activity status of Ras. The assay optimization showed that only 60 nM concentration of purified H-Ras protein was needed. The functionality of the assay was proved by detecting the effect of H-Ras guanine nucleotide exchange factor, Son of Sevenless. The signal-to-background ratio up to 7.7 was achieved with an average assay coefficient of variation of 9.1%. The use of a low concentration of purified protein is desirable and the signal-to-background ratio of 3.4 was achieved in the assay at a concentration of 60 nM for H-Ras and SOS proteins. The need of only one labeled molecule and the ability to decrease the quantities of purified proteins used in the experiments are valuable qualities in HTS showing the potential of the QRET method.
Ras 是一种小 GTP 结合蛋白，其突变在肿瘤中很常见，Ras 癌基因已被确定参与癌症的早期发生。Ras 活性的检测对于确定控制细胞生长和分化的细胞信号事件至关重要。因此，开发改进的方法来筛选针对小 GTPase 或其调节剂及其信号通路的新型潜在药物非常重要。已经开发了几种用于小 GTPase 研究的测定方法，但所有这些方法都存在限制，不适合高通量筛选 (HTS) 使用。包括分离、使用放射性标记物或耗时的免疫印迹以及需要大量纯化蛋白在内的多个步骤降低了这些方法的易用性。在这里，我们开发了一种基于单标记利用均相猝灭共振能量转移技术 (QRET) 的均质 H-Ras 活性测定法。在 QRET 方法中，铽标记的 GTP (Tb-GTP) 与小 GTPase 蛋白 H-Ras 的结合保护了标记物的信号免受猝灭，而 Tb-GTP 的未结合部分的信号则被可溶性猝灭剂猝灭。这使得能够快速确定 Ras 活性状态的变化。通过检测 Son of Sevenless（一种 Ras 鸟苷核苷酸交换因子）对 Ras 的影响，证明了测定法的功能。实现了高达 7.7 的信号与背景比，平均测定变异系数为 9.1%。使用低浓度的纯化蛋白是理想的，在 60 nM 的浓度下，H-Ras 和 SOS 蛋白的测定中实现了 3.4 的信号与背景比。仅使用一个标记分子的能力以及在实验中减少使用的纯化蛋白的数量是 HTS 的有价值的特性，表明了 QRET 方法的潜力。
