High Throughput Methods for Single Cell Proteomics

单细胞蛋白质组学的高通量方法

基本信息

批准号：
10609071
负责人：
John R Yates III
金额：
$ 22.63万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10609071
关键词：
3-Dimensional Action Potentials Adsorption Alzheimer&apos 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 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 transcriptome sequencing

项目摘要

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/摘要为了了解疾病带来的神经元和神经元电路正常功能的出发，我们将使用贴片夹具开发方法来测量单个神经元的电生理学和然后使用基于质谱的蛋白质组学来测量蛋白质组。现在神经元可以由具有疾病基因的患者的皮肤细胞创建，以确定表型的基因型遗传缺陷与电生理学之间的关系，我们开发的方法具有很大的显着提高我们研究大脑疾病的能力的潜力。 “大脑”类器官是还可以从皮肤细胞创建以概括神经元的3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与神经元和其他细胞的扰动如何与分子表型有关。单细胞RNA-SEQ用于测量神经元中的基因表达，但是基因表达曲线无法解释蛋白质合成的速率，降解，蛋白质，后翻译修饰和酶促活性，所有这些都是关键的细胞功能由蛋白质。单细胞质谱法已应用于神经元，以测量代谢产物和神经肽，但测量蛋白质的努力滞后。我们已经确定蛋白质可以在电生理学后在神经元中测量，但在这里我们建议大大增加测量和吞吐量。这些方法将广泛适用于补丁夹技术是一种广泛使用的技术，用于测量各种细胞类型的离子电流神经元，心肌细胞，肌肉纤维和胰腺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理致神经元