Microfluidic Chip for the Analysis of Cell-Surface Proteins

细胞表面蛋白质在细胞信号传导、细胞间通讯以及细胞间、病原体和病原体之间的相互作用中发挥重要作用。 免疫识别事件。它们作为指示疾病的标志物或有吸引力的治疗靶点的价值 干预措施早已得到认可。小型化设备已经成为研究一种 各种细胞内和细胞外信号传导过程，读数依赖于活细胞成像或 免疫细胞化学，并因此，对高特异性抗体的可用性和/或需要 荧光报告基因。蛋白质组学技术能够提供来自全细胞的全面数据， 文化，但与蛋白质定位，丰度和翻译后的存在相关的信息， 修饰、蛋白质同种型或蛋白质突变经常丢失。 为了解决这些局限性，本提案的目标是开发第一个平台，该平台将联合收割机 微流体的细胞处理能力与质谱（MS）检测的力量，以推进 这项技术可以追踪细胞表面蛋白质组对各种刺激的反应。目标1将侧重于 开发一个芯片实验室平台，使细胞捕获，刺激，细胞的酶促释放， 表面蛋白，酶消化和蛋白受体和抗原的明确的基于MS的鉴定， 包括它们的同种型和可能的突变序列。目标2将重点关注设备的优化， 实现芯片上装载的细胞的最佳刺激条件，实现用于 细胞表面受体蛋白，并开发定量方法评估丰度的变化， 这些受体。目标3将重点展示用于细胞表面识别的微流控装置 参与EGFR信号传导的GPCR反式激活和细胞周期过程的启动的过程 支持Her 2+癌细胞的增殖。与人类健康的相关性。所提出的微流体 技术将有助于观察受体激活/反式激活和细胞重塑- 表面蛋白质组揭示新的细胞信号起始机制和开发治疗的前景 与常规药物靶向协同作用的策略。它还将使识别细胞表面 具有生物标记潜力的抗原。

Cell-surface proteins play a major role in cell signaling, inter-cellular communication, and cell-cell, pathogen and immune recognition events. Their value as markers indicative of disease or attractive targets for therapeutic interventions has been long recognized. Miniaturized devices have emerged as useful platforms for studying a variety of intra and extra-cellular signaling processes, the read-out relying on live-cell imaging or immunocytochemistry, and, therefore, on the availability of high-specificity antibodies and/or the need for fluorescent reporters. Proteomic technologies are capable of delivering comprehensive data from whole-cell cultures, but information related to protein localization, abundance, and the presence of posttranslational modifications, protein isoforms or protein mutations is often lost. To address these limitations, the objective of this proposal is to develop the first platform that will combine the cell-handling capabilities of microfluidics with the power of mass spectrometry (MS) detection to advance a technology that can track changes in the cell-surface proteome in response to various stimuli. Aim 1 will focus on the development of a lab-on-a-chip platform that will enable cell capture, stimulation, enzymatic release of cell- surface proteins, enzymatic digestion and unambiguous MS-based identification of protein receptors and antigens, including their isoforms and possible mutated sequences. Aim 2 will focus on the optimization of the device for achieving optimal stimulation conditions of cells loaded on the chip, achieving the necessary detection limits for cell-surface receptor proteins, and developing a quantitative approach for assessing changes in the abundance of these receptors. Aim 3 will focus on demonstrating the microfluidic device for the identification of cell-surface processes that are involved in GPCR transactivation of EGFR signaling, and the initiation of cell cycle processes that support the proliferation of Her2+ cancer cells. Relevance to human health. The proposed microfluidic technology will facilitate the observation of receptor activation/transactivation and the remodeling of the cell- surface proteome to reveal new cell signal initiation mechanisms and prospects for developing treatment strategies that work in synergy with conventional drug targeting. It will also enable the identification of cell-surface antigens with biomarker potential.