High Throughput Methods for Single Cell Proteomics

项目摘要/摘要 为了了解疾病引起的神经元正常功能和神经元回路的偏差，我们 将开发使用膜片钳来测量单个神经元的电生理学的方法，并 然后使用基于质谱学的蛋白质组学来测量蛋白质组。现在神经元可以 从携带疾病基因的患者的皮肤细胞中产生，以确定基因型和表型 遗传缺陷与电生理的关系，我们开发的方法有很大的优势 有可能显著提高我们研究人类大脑疾病的能力。“大脑”有机化合物是 也是从皮肤细胞创建的，以概括神经元的三维环境，并包括 兴奋性和抑制性神经元。同时测量电生理学和蛋白质的能力 表达将允许确定疾病对神经元和其他细胞的扰动程度 与分子表型有关。单细胞RNA-SEQ用于测量神经元中的基因表达， 但基因表达谱不能解释蛋白质合成、降解、蛋白稳定、后 翻译修饰和酶活性，所有这些都是细胞完成的关键功能 通过蛋白质。单细胞质谱仪已被应用于神经元，以测量代谢物和 神经肽，但测量蛋白质组的努力一直滞后。我们已经确定蛋白质可以 在电生理学之后在神经元中被测量，但在这里我们建议大大增加 测量以及吞吐量。这些方法将广泛应用于膜片钳技术 技术是一种广泛使用的技术，用于测量各种电池类型中的离子电流，包括 神经元、心肌细胞、肌肉纤维和胰岛β细胞。此外，这些方法将 使实验能够确定恢复正常电生理的药物的作用机制 到神经元

PROJECT SUMMARY/ ABSTRACT To understand deviations in normal function of neurons and neuronal circuits brought on by disease, we will develop methods using patch clamping to measure the electrophysiology of individual neurons and then use mass spectrometry-based proteomics to measure the proteome. Now that neurons can be created from the skin cells of patients harboring disease genes to determine genotype to phenotype relationships between the genetic defects and electrophysiology, the methods we develop have great potential to significantly improve our ability to study human diseases of the brain. “Brain” organoids are also being created from skin cells to recapitulate a 3-dimensional environment for neurons and to include excitatory and inhibitory neurons. The ability to concurrently measure electrophysiology and protein expression will allow a determination of how disease related perturbations to neurons and other cells are related to molecular phenotypes. Single cell RNA-SEQ is used to measure gene expression in neurons, but gene expression profiles fail to account for rates of protein synthesis, degradation, proteostasis, post translational modification and enzymatic activity, all of which are critical cellular functions accomplished by proteins. Single cell mass spectrometry has been applied to neurons to measure metabolites and neuropeptides, but efforts to measure the proteome have lagged. We have established that proteins can be measured in neurons after electrophysiology, but here we propose to greatly increase the scale of measurements as well as the throughput. These methods will be broadly applicable as patch clamping techniques is a widely used technique to measure ionic currents in a variety of cell types including neurons, cardiomyocytes, muscle fibers and pancreatic beta cells. Furthermore, these methods will enable experiments to determine the mechanism of action of drugs that restore normal electrophysiology to neurons