High Throughput Measurement of Cellular Signaling

描述(由申请人提供)：我们将开发自动化微流控设备，用于高通量测量人类细胞中的激酶激活。这些设备最终将能够通过测量和比较大量单个细胞中多个不同的激酶的活性来检测和表征细胞中信号转导通路的状态。这些设备将自动执行高通量测量所需的所有处理和测量步骤，包括细胞传输、将激酶底物多肽加载到细胞中、细胞裂解以及释放的多肽的分离和检测。磷酸化底物和非磷酸化底物的比率将被用来衡量每个细胞中所检测的每一种激酶的激活程度。激酶在细胞生理学的几乎所有方面都扮演着重要的角色，也是密集的基础和制药科学研究的焦点，因此这些测量在生物医学科学中具有广泛的适用性。此外，由于群体中单个细胞之间细胞行为的异质性，高通量单细胞分析对于了解信号网络如何相互作用来控制细胞生理至关重要。因此，开发一种能够确定单细胞中激酶的数量、身份和激活程度的技术将极大地促进我们对细胞信号在健康和疾病中的分子机制的理解。

DESCRIPTION (provided by applicant): We will develop automated microfluidic devices for the high throughput measurements of kinase activation in human cells. These devices will ultimately be capable of detecting and characterizing the states of signal transduction pathways in cells by measuring and comparing the activity of multiple different kinases simultaneously in a large number of individual cells. The devices will automatically perform all of the processing and measurement steps required for high throughput measurements including cell transport, loading of kinase substrate peptides into the cells, cell lysis, and the separation and detection of the released peptides. The ratios of phosphorylated to nonphosphorylated substrates will be used as a measure of the degree of activation of each kinase assayed in each cell. Kinases play crucial roles in virtually all aspects of cellular physiology and are the focus of intense basic and pharmaceutical science research, thus these measurements have widespread applicability in the biomedical sciences. Moreover, by virtue of the heterogeneity of cellular behavior among individual cells in a population, high throughput single cell assays are critical for understanding how signaling networks interact to control cell physiology. Developing a technology, therefore, that is capable of determining the number, identities, and degree of activation of kinases in single cells will substantially advance our understanding of the molecular mechanisms of cellular signaling in health and disease.