High Throughput Measurement of Cellular Signaling

描述（由申请人提供）：我们将开发用于人类细胞中激酶激活的高通量测量的自动化微流体装置。这些装置最终将能够通过同时测量和比较大量单个细胞中多种不同激酶的活性来检测和表征细胞中信号转导途径的状态。该设备将自动执行高通量测量所需的所有处理和测量步骤，包括细胞运输，将激酶底物肽加载到细胞中，细胞裂解以及释放肽的分离和检测。磷酸化与非磷酸化底物的比率将被用作每个细胞中测定的每个激酶活化程度的量度。激酶几乎在细胞生理学的所有方面都起着至关重要的作用，是基础科学和制药科学研究的重点，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行为的异质性，高通量单细胞分析对于理解信号网络如何相互作用以控制细胞生理至关重要。因此，开发一种能够确定单个细胞中激酶的数量、身份和激活程度的技术，将大大促进我们对健康和疾病中细胞信号传导的分子机制的理解。

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.