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: 10709859
• 负责人: Vilmos Kertesz
• 金额: $ 22.84万
• 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/10709859
• 关键词: Agreement; Amiodarone; Anti-Arrhythmia Agents; Area; BT 474; Basic Science; Biomedical Research; Calibration; Cell Line; Cell physiology; Cells; Cellular Metabolic Process; Chemicals; Communities; Complex; Custom; Cytolysis; Data; Drug Kinetics; Drug Targeting; Electrospray Ionization; Environment; 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; 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; Toxicology; advanced disease; cell type; cellular development; computer framework; disease diagnosis; electric field; high reward; high risk; improved; insight; interest; ionization; nano-electrospray; novel; pharmacodynamic model; pharmacologic; response; single cell analysis; single molecule; small molecule; 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影响的基本认识 对刺激的反应几乎影响到生物医学研究的方方面面，包括疾病诊断， 治疗和预防。细胞代谢是天然的异质性的，预测细胞对 刺激将取决于细胞内代谢物浓度在整个细胞群体中的分布。然而， 然而，对于小分子和治疗药物来说，直接测量细胞内浓度的经验很少。 许多药物以细胞内为靶点，需要在特定浓度下到达作用部位 有效。它们在细胞群体中的数量分布是目前无法测量的关键变量 了解多种药物的药理和毒理作用。 解开异质细胞反应的最好方法是单独分析种群中的每个细胞。 不幸的是，目前用于测量小分子的单细胞分析方法缺乏许多关键的 进行常规分析所需的特征。理想的单细胞表征方法必须是定量的、快速的、 坚固、灵敏，适用于多种细胞类型，能够测量多种分子。匮乏 限制了对支撑异构性的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本信息，如 国家普通医学科学研究所(NIGMS)使命的一部分。 这项研究的目标是评估一种高风险/高回报的方法，脉冲单细胞团 光谱(p-SC-MS)，一种捕获、裂解和分析单个细胞的新概念，具有显著的 与现有的小分子单细胞分析方法相比，提高了灵敏度和吞吐量。我们的 这种方法需要克服小范围和低成本的液滴捕获和细胞裂解的高风险挑战 高电场环境。这种风险被相应的较高回报所抵消：大幅增加 灵敏度(&gt；50,000倍)、单细胞分析吞吐量(&gt；6倍)和细胞介质容差 艺术。该平台易于使用，并将通过三个具体的 旨在解决这一概念的可行性。具体目标#1：演示按需捕获单细胞液滴 以及使用定制的脉冲纳米电喷雾电离源提取代谢物。具体目标2：验证 用胺碘酮和N-脱乙基胺碘酮测定p-SC-MS的定量准确性 (NDEA)。具体目标#3：演示代谢物和脂类的测量和比较 在HeLa、HepG2、MD-AMB、BT-474和OK-F6细胞系中进行复合培养。这项研究的最终成果将是 是一种新的、高影响力的技术，能够对单个细胞中的小分子进行化学表征 与目前的技术相比，灵敏度提高了50,000倍，速度提高了6倍，实现了 细胞内分子分布的分辨率。