Development of Single Cell Printing-Liquid Vortex Capture-Mass Spectrometry for the Metabolic Profiling of Single Cells

用于单细胞代谢分析的单细胞打印-液体涡旋捕获-质谱法的开发

• 批准号: 10339794
• 负责人: John F Cahill
• 金额: $ 37.45万
• 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 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10339794
• 关键词: Address; Agreement; Area; Biological; Blood capillaries; Caliber; Cell Cycle; Cell Nucleus; Cell Separation; Cells; Cellular Morphology; Cellular Structures; Chemicals; Chemistry; Chloroquine; Communities; Cytolysis; Dactinomycin; Data; Detection; Development; Disease; Dyes; Ensure; Exposure to; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Fluorescent Dyes; Goals; Growth; Hela Cells; HepG2; High Pressure Liquid Chromatography; Individual; Label; Link; Lipids; Liquid substance; Lysosomes; Mammalian Cell; Mass Chromatography; Mass Spectrum Analysis; Measurement; Measures; Medicine; Metabolic; Methods; Morphologic artifacts; Nutrient; Optics; Organ; Pharmaceutical Preparations; Phenotype; Population; Printing; Process; Proteins; Research; Rhodamine 123; Sampling; Solvents; Stress; System; Techniques; Technology; Therapeutic; Time; Validation; Work; dalton; fluorescence imaging; high throughput analysis; imaging capabilities; imaging system; improved; insight; metabolomics; new technology; protein expression; response; single cell technology; single molecule; small molecule; success; tool; treatment response

Project Summary The mechanisms underpinning heterogeneous metabolic response between individual cells remain poorly understood, though such mechanisms have ramifications across nearly all of medicine, e.g., disease detection, response to stress, treatment, and more. Intracellular small molecule metabolite, lipid and therapeutic concentrations are often inferred through protein expression or directly measured as the average of 1000’s of cells at once, providing only a population-level view of heterogeneous metabolic response. This obfuscates differentiation of anomalous metabolic cell phenotypes and small molecule associations occurring within cells. Currently there exists a gap in analytical technologies for the measure of small molecules (<1000 Dalton) in single mammalian cells. Most technologies lack the sensitivity, broad chemical coverage, quantitative capability, and/or the sampling throughput needed to enable the mechanisms of heterogeneous metabolic response of cells to be explored, but recently the development of single cell printing-liquid vortex capture-mass spectrometry (SCP-LVC-MS) has overcome many of these limitations. We hypothesize that this technology could resolve the current analytical gap in the quantitative measure of small molecules from single cells. The goal of this research is to address key technical challenges regarding validation of the quantitative accuracy of SCP-LVC-MS (Aim #1), representativeness of SCP-LVC-MS measured cellular chemical profiles (Aim #2), and ability to relate SCP-LVC-MS measurements to established single-cell technologies (Aim #3) so that SCP-LVC-MS can become an effective research tool in the biomedical community and enable associations between metabolites and therapeutics to be investigated on an individual cellular basis for the first time. These technical challenges are addressed by measure of localized intracellular compounds and comparison with in- capillary fluorescence measurements, by validating the representativeness of SCP-LVC-MS measured chemical profiles through comparison of spectra acquired before and after single cell isolation and by incorporating fluorescence microscopy into the SCP-LVC-MS optical detection and cell isolation system. These aims work together to validate and improve the capabilities of SCP-LVC-MS to answer questions regarding the central challenge of understanding the cellular mechanisms underpinning heterogeneous metabolic response, by filling in the technological gap that exists in this area for quantitative, high throughput analysis of metabolites, lipids and drugs in single cells. The completion of this research will enable fundamental biomedical questions to be pursued that could not be before, such as how intracellular metabolite concentrations relate to each other or how expression of a protein quantitatively impacts therapeutic response in the same cell. Fundamental questions like these have impacts in how we understand disease, how single-cell chemistry links across biological scales (e.g., organ level phenotypes), and many other fundamental aspects of medicine.

项目概要 支持单个细胞之间异质代谢反应的机制仍然很差 众所周知，尽管这种机制对几乎所有医学领域都有影响，例如疾病检测、 对压力、治疗等的反应。 浓度通常通过蛋白质表达推断或直接测量为 1000 个蛋白质的平均值 细胞一次，仅提供异质代谢反应的群体水平视图。 细胞内发生的异常代谢细胞表型和小分子关联的分化。 目前，在测量小分子（<1000 道尔顿）的分析技术方面存在差距。 大多数技术缺乏敏感性、广泛的化学覆盖范围、定量能力、 和/或实现细胞异质代谢反应机制所需的采样通量 有待探索，但最近单细胞打印-液体涡旋捕获-质谱技术的发展 (SCP-LVC-MS) 克服​​了许多这些限制，我们发现这项技术可以解决这个问题。 目前在单细胞小分子定量测量方面存在分析差距。 本研究的目标是解决有关定量验证的关键技术挑战 SCP-LVC-MS 的准确性（目标 #1）、SCP-LVC-MS 测量的细胞化学特征的代表性 （目标 #2），以及将 SCP-LVC-MS 测量与已建立的单细胞技术相关联的能力（目标 #3），以便 SCP-LVC-MS 可以成为生物医学界和促进协会的有效研究工具 首次在个体细胞基础上研究代谢物和治疗方法之间的关系。 通过测量局部细胞内化合物并与内部比较来解决技术挑战 通过验证 SCP-LVC-MS 测量的化学物质的代表性来进行毛细管荧光测量 通过比较单细胞分离前后获得的光谱并结合 荧光显微镜进入 SCP-LVC-MS 光学检测和细胞分离系统。 这些目标共同验证和提高 SCP-LVC-MS 回答问题的能力 关于理解支持异质性的细胞机制的核心挑战 代谢反应，通过填补该领域存在的定量、高通量技术空白 这项研究的完成将为单细胞中代谢物、脂质和药物的分析奠定基础。 以前无法解决的生物医学问题，例如细胞内代谢物浓度如何 彼此相关，或者蛋白质的表达如何定量地影响同一细胞中的治疗反应。 诸如此类的基本问题会影响我们如何理解疾病以及单细胞化学如何联系 跨生物尺度（例如器官水平表型）以及医学的许多其他基本方面。