High Throughput Methods for Single Cell Proteomics

单细胞蛋白质组学的高通量方法

• 批准号: 10433158
• 负责人: John R Yates III
• 金额: $ 26.63万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10433158
• 关键词: 3-Dimensional; Action Potentials; Adsorption; Alzheimer' s Disease; Biochemical; Brain; Brain Diseases; Cardiac Myocytes; Cell Size; Cell physiology; Cells; DNA Sequence Alteration; Disease; Electric Capacitance; Electrodes; Electrophysiology (science); Environment; Gene Expression; Genes; Genetic; Genotype; Goals; In Situ; Individual; Mass Spectrum Analysis; Measurement; Measures; Membrane Potentials; Mental Depression; Methods; Molecular; Muscle Fibers; Mutation; Neurons; Neuropeptides; Organoids; Outcome; Patch-Clamp Techniques; Pathway interactions; Patients; Phenotype; Post-Translational Protein Processing; Potassium; Protein Biosynthesis; Proteins; Proteome; Proteomics; Protocols documentation; Research; Rest; Sampling; Schizophrenia; Signal Transduction; Skin; Sodium; Structure of beta Cell of islet; Techniques; autism spectrum disorder; base; cell type; drug action; excitatory neuron; experimental study; human disease; improved; inhibitory neuron; mass spectrometer; molecular phenotype; nanolitre; nervous system disorder; neuronal circuitry; neurotransmitter release; patch clamp; pressure; programs; protein expression; proteostasis; single cell analysis; single-cell RNA sequencing

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

项目摘要/摘要 为了了解疾病引起的神经元和神经回路正常功能的偏差，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， 然后使用基于质谱的蛋白质组学来测量蛋白质组。既然神经元可以 从携带疾病基因的患者的皮肤细胞中产生，以确定基因型与表型 遗传缺陷和电生理学之间的关系，我们开发的方法有很大的 有可能大大提高我们研究人类大脑疾病的能力。“大脑”类器官是 也是由皮肤细胞产生的，以重现神经元的三维环境， 兴奋和抑制神经元。同时测量电生理和蛋白质的能力 表达将允许确定神经元和其它细胞的疾病相关扰动如何被 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、蛋白质稳态、后 翻译修饰和酶活性，所有这些都是完成的关键细胞功能 通过蛋白质。单细胞质谱法已经应用于神经元以测量代谢物， 神经肽，但测量蛋白质组的努力已经滞后。我们已经确定蛋白质可以 在电生理学之后在神经元中测量，但是在这里，我们建议大大增加 测量以及吞吐量。这些方法将广泛适用于膜片钳 技术是一种广泛用于测量多种细胞类型中离子电流的技术， 神经元、心肌细胞、肌纤维和胰腺β细胞。此外，这些方法将 使实验能够确定恢复正常电生理学的药物的作用机制 对神经元