Novel tools for protein phosphorylation analysis based on proton release detection
Novel tools for protein phosphorylation analysis based on proton release detection
复制标题
基于质子释放检测的蛋白质磷酸化分析新工具
DOI：
--
复制
发表时间：
2014
期刊：
影响因子：
--
通讯作者：
P. Estrela
中科院分区：
文献类型：
--
作者：
Nikhil Bhalla;M. D. Lorenzo;G. Pula;P. Estrela
磷酸化是真核细胞中蛋白质翻译后最重要的修饰，它是由激酶催化的。蛋白质磷酸化和去磷酸化之间的平衡对生理过程的调节至关重要，其不平衡是许多疾病的原因。无标签生物传感技术可以为高通量药物发现平台提供改进的设备。我们开发了两种通用的方法来检测与激酶[1]催化的蛋白质磷酸化相关的质子（H+）的释放。第一种方法是基于氧化物半导体界面的pH敏感响应，第二种方法是使用商业微pH电极检测磷酸化反应中的pH变化。在ph敏感的氮化硅上检测（图1）显示出非常高的灵敏度和响应。我们的假设和实验结果之间有很好的相关性。以PKC-α激酶磷酸化髓鞘碱性蛋白为例，氮化硅基电解质绝缘体半导体电容器结构在磷酸化后的栅极电压变化为37 mV，而在抑制激酶活性的对照实验中栅极电压变化小于2 mV。商用微pH计检测到的pH变化很小，与在Si3N4上观察到的pH变化相比，pH变化约为1.5%。这是因为由于溶液的缓冲能力和开发稳定的微pH电极的技术困难，直接检测pH变化具有挑战性。然而，通过统计分析证明，它仍然能够显著区分磷酸化和非磷酸化样品。这些技术可以很容易地用于多路复用阵列和激酶活性的高通量分析，这将代表生物医学研究和药物发现的重要创新。
Phosphorylation is the most important post-translational modification of proteins in eukaryotic cells and it is catalysed by enzymes called kinases. The balance between protein phosphorylation and dephosphorylation is critical for the regulation of physiological processes and its unbalance is the cause of several diseases. Label-free biosensing techniques can provide improved devices for high throughput drug discovery platforms. We have developed two versatile methods to detect the release of protons (H+) associated with the protein phosphorylation catalysed by kinases [1]. The first approach is based on the pH-sensitive response of oxide semiconductor interfaces and the second method detects the pH changes in phosphorylation reaction using a commercial micro pH electrode. Detection on pH-sensitive silicon nitride (Figure 1) shows a remarkably high sensitivity and response. A good correlation was observed between our hypothesis and the experimental results. Using phosphorylation of myelin basic protein by PKC-α kinase as a case study, silicon nitride based electrolyte insulator semiconductor capacitor structures revealed a change in gate voltage of 37 mV upon phosphorylation as compared to less than 2 mV in control experiments wherein kinase activity was inhibited. The commercial micro-pH meter detected a low change, at around 1.5% of change in pH as compared to the change in pH observed on Si3N4. This is because direct detection of pH variations is challenging due to the buffering capabilities of the solution and the technological difficulties in developing stable micro-pH electrodes. Nevertheless it is still able to significantly distinguish between the phosphorylated and non-phosphorylated sample proved by statistical analysis. These techniques can be readily adopted for multiplexed arrays and high throughput analysis of kinase activity, which will represent an important innovation in biomedical research and drug discovery.