A Microfluidic/Nanofluidic System for Rapid Single Cell Proteomics

蛋白质组学通常涉及对大量细胞的蛋白质成分进行分析。差一点 细胞的所有机器都是蛋白质，所以许多疾病以及对刺激和应激源的反应都是蛋白质 主要表现在蛋白质水平上。由于许多原因，蛋白质组正面临着完全表征的挑战。一些人 蛋白质组的关键属性使其特征如此具有挑战性的是：大量的 在单个细胞中表达的蛋白质，蛋白质相对丰度的宽动态范围，以及 许多修饰可以在活性和非活性状态之间切换蛋白质。进一步增加 复杂性，在细胞水平上存在显著的异质性，这是通过整体分析平均出来的。至 更好地理解细胞异质性的生物学和健康影响，单细胞蛋白质组学 出现了。然而，作为单细胞技术，需要改进使能分析技术。 蛋白质组学增加了极小样本量的额外挑战。此应用程序侧重于 用于改进单细胞蛋白质组的新型电动微流控/纳米流控系统的研制 分析表明，这与目前的超高效液相色谱有很大的偏差 (UPLC)-基于质谱仪(MS)的方法。提出的电动微流控/纳米流控 预计系统将从以下方面提供改进的性能：减少样本损失，增加样本 浓度洗脱进入质谱仪，减少多肽污染，效率高 分离和快速分析。本申请的目的主要集中在：1)制造和优化 关键功能模块；2)将这些功能模块集成到一个完整的系统中； 测量整个系统的性能。系统性能的主要指标 将是分析的速度和确定的蛋白质组的数量。如果成功，这将是 应用将为单细胞蛋白质组学提供一种新的系统，将提高分析速度 而不牺牲蛋白质组覆盖的深度。

Proteomics typically involves the analysis of the protein components of large populations of cells. Nearly all the of a cell’s machinery are proteins, so many diseases and responses to stimuli and stressors are manifested at the protein level. Proteomes are challenging to characterize fully for many reasons. Some of the key attributes of a proteome that make its characterization so challenging are: the large number of proteins expressed in a single cell, the wide dynamic range of protein relative abundance, and the numerous modifications that can switch proteins between active an inactive states. To further increase complexity, significant heterogeneity exists at the cellular level that is averaged out by bulk analyses. To better understand biological and health implications of cellular heterogeneity, single cell proteomics has emerged. However, improvements in the enabling analytical technologies are needed as single-cell proteomics adds the additional challenge of extremely small sample size. This application focuses on the development of a novel electrokinetic microfluidic/nanofluidic system for improved single-cell proteome analyses that is a significant deviation from the current ultra-performance liquid chromatography (UPLC)-mass spectrometry (MS) based approaches. The proposed electrokinetic microfluidic/nanofluidic system is expected to offer improved performance from: reduced sample loss, increased sample concentration eluted into the mass spectrometer, reduced peptide contamination, high efficiency separations, and rapid analyses. The Aims of this application focus on: 1) fabrication and optimization of the key functional modules, and 2) integration of these functional modules into a complete system and measurement of the performance of the complete system. The primary metrics for system performance will be the speed of the analysis and the number of protein groups identified. If successful, this application will provide a novel system for single-cell proteomics that will improve the analysis speed without sacrificing depth of proteome coverage.