Quantitative Single Cell Mass Spectrometry for Small Molecules with Unprecedented Sensitivity

具有前所未有的灵敏度的小分子定量单细胞质谱分析

• 批准号: 10431204
• 负责人: Vilmos Kertesz
• 金额: $ 22.34万
• 依托单位: UT-BATTELLE, LLC-OAK RIDGE NATIONAL LAB
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-24 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10431204
• 关键词: Agreement; Amiodarone; Area; Arts; BT 474; Basic Science; Biomedical Research; Calibration; Cell Line; Cell physiology; Cells; Cellular Metabolic Process; Chemicals; Communities; Complex; Custom; Cytolysis; Data; Diethylnitrosamine; Drug Kinetics; Drug Targeting; Environment; Foundations; Goals; Heart; Hela Cells; HepG2; Heterogeneity; Individual; Investigation; Ions; Isotope Labeling; Lipids; Mass Spectrum Analysis; Measures; Metabolic; Metabolism; Methodology; Methods; Mission; Molecular; Morphologic artifacts; National Institute of General Medical Sciences; Pharmaceutical Preparations; Pharmacology and Toxicology; Physiologic pulse; Population; Prevention; Primary carcinoma of the liver cells; Propranolol; Pythons; Research; Resolution; Risk; Sampling; Site; Solvents; Source; Speed; Statistical Data Interpretation; Stimulus; System; Techniques; Technology; Testing; Therapeutic; advanced disease; base; cell type; cellular development; computer framework; disease diagnosis; electric field; high reward; high risk; improved; insight; interest; ionization; nano-electrospray; novel; pharmacodynamic model; response; single cell analysis; single molecule; small molecule; small molecule therapeutics; stressor; tool

Project Summary Fundamental understanding of the development of cellular metabolic heterogeneity and its impact on cell response to stimuli has ramifications on nearly every aspect of biomedical research including disease diagnosis, treatment and prevention. Cellular metabolism is naturally heterogeneous and prediction of cellular response to stimuli will depend on the distribution of intracellular metabolite concentrations across a cell population. However, for small molecules and therapeutics direct empirical measure of intracellular concentrations is rare, though many drugs target inside the cell and require reaching their site of action at a specific concentration to be effective. Their quantitative distribution in a cell population is a key, currently unmeasurable, variable to understanding many drug’s pharmacological and toxicological effects. The best way to deconvolute heterogeneous cell response is to individually profile each cell in the population. Unfortunately, current single cell analysis approaches for measuring small molecules lack a number of critical features needed to make analysis routine. An ideal single cell characterization method must be quantitative, fast, robust, sensitive, applicable to a range of cell types and be able to measure a wide range of molecules. The lack of such a system restricts detailed investigations into the fundamental mechanisms underpinning heterogeneous cell response; foundational information needed for advancing disease diagnosis, treatment and prevention as part of the mission of National Institute of General Medical Sciences (NIGMS). The goal of this research is to evaluate a high-risk/high-reward approach, pulsed single cell mass spectrometry (p-SC-MS), a novel concept of capturing, lysing and analyzing single cells with significantly improved sensitivity and throughput over existing single cell analysis methodologies for small molecules. Our approach requires overcoming high-risk challenges in droplet capture and cell lysis at small scale and under a high electric field environment. This risk is offset by a correspondingly high reward: massively increased sensitivity (>50,000X), single cell analysis throughput (>6X) and cell media tolerance over the current state-of- the-art. The platform is easy to use and will be shown to be applicable for many cell types through three specific aims tackling the feasibility of the concept. Specific Aim #1: Demonstrate single cell droplet-on-demand capture and metabolite extraction using a custom pulsed-nanoelectrospray ionization source. Specific Aim #2: Validate the quantitative accuracy of p-SC-MS through measure of amiodarone (AMIO) and N-desethylamiodarone (NDEA) in single HepG2 cells. Specific Aim #3: Demonstrate measure and comparison of metabolites and lipids in HeLa, HepG2, MD-AMB, BT-474 and OK-F6 cell lines in complex media. The end product of this research will be a novel, high-impact technology that enables chemical characterization of small molecules from single cells with a massive >50,000X improvement in sensitivity and 6X improvement in speed over current art, enabling resolution of intracellular molecular distributions.

项目摘要 对细胞代谢异质性发展及其对细胞的影响的基本了解 对刺激的反应几乎对生物医学研究的各个方面都有影响，包括疾病诊断， 治疗和预防。细胞代谢自然是异质的，并且预测了细胞对 刺激将取决于细胞内代谢物浓度在细胞种群中的分布。然而， 对于小分子和治疗直接对细胞内浓度的直接经验测量很少见，但是 许多药物靶向细胞内，需要以特定浓度到达其作用部位 有效的。它们在细胞种群中的定量分布是关键，目前无法衡量，可变为 了解许多药物的药物和毒理学作用。 反应异质细胞反应的最佳方法是单独介绍人群中的每个细胞。 不幸的是，当前测量小分子的单细胞分析方法缺乏许多关键 进行分析常规所需的功能。理想的单细胞表征方法必须是定量的，快的， 健壮，敏感，适用于各种细胞类型，并能够测量各种分子。缺乏 这样的系统限制了对基本机制的详细研究 细胞反应；推进疾病诊断，治疗和预防所需的基础信息 美国国家一般医学科学研究所（NIGMS）任务的一部分。 这项研究的目的是评估高风险/高回报方法，脉冲单细胞质量 光谱法（P-SC-MS）是一种新颖的捕获，裂解和分析单细胞的新概念 对小分子的现有单细胞分析方法提高了灵敏度和吞吐量。我们的 方法需要克服小规模和小细胞裂解和细胞裂解的高风险挑战 高电场环境。这种风险被相应高的奖励所抵消：大量增加 灵敏度（> 50,000x），单细胞分析吞吐量（> 6倍）和细胞介质的耐受性 艺术。该平台易于使用，并将显示通过三种特定的细胞类型适用 目的解决该概念的可行性。特定目标＃1：演示单细胞液滴按需捕获 并使用自定义的脉冲纳米电源电离源代谢物提取。特定目标＃2：验证 P-SC-MS的定量准确性通过测量amidarone（AMIO）和N-甲基氨基二聚体的测量 （NDEA）在单个HEPG2细胞中。特定目的＃3：演示代谢物和脂质的测量和比较 在HELA，HEPG2，MD-AMB，BT-474和OK-F6细胞系中的复杂介质中。这项研究的最终产品将 成为一种新颖的高影响力技术，可以使单个细胞的小分子化学表征 敏感性> 50,000倍提高，速度比当前艺术提高了6倍 细胞内分布的分辨率。