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

项目摘要 支持个体细胞间异质性代谢反应的机制仍然很差 理解，虽然这样的机制在几乎所有的医学中都有分支，例如，疾病检测， 应对压力、治疗等。细胞内小分子代谢物、脂质和治疗剂 浓度通常通过蛋白质表达来推断，或者直接测量为1000个蛋白质的平均值。 细胞，仅提供异质性代谢反应的群体水平视图。这使人困惑 异常代谢细胞表型的分化和细胞内发生的小分子缔合。 目前，在用于测量小分子（<1000道尔顿）的分析技术方面存在差距， 单个哺乳动物细胞。大多数技术缺乏敏感性、广泛的化学覆盖面、定量能力， 和/或实现细胞的异质代谢反应机制所需的采样通量 但最近单细胞打印-液体涡旋捕获-质谱的发展 （SCP-LVC-MS）已经克服了这些限制中的许多。我们假设这项技术可以解决 目前单细胞小分子定量测量的分析空白。 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的准确度（目标1），SCP-LVC-MS测定的细胞化学特征的代表性 (Aim#2），以及将SCP-LVC-MS测量与已建立的单小区技术相关联的能力（目标#3）， SCP-LVC-MS可以成为生物医学界的有效研究工具， 代谢物和治疗剂之间的关系将首次在单个细胞的基础上进行研究。这些 技术挑战是通过测量局部细胞内化合物和与in- 毛细管荧光测量，通过验证SCP-LVC-MS测量化学品的代表性 通过比较在单细胞分离之前和之后获得的光谱以及通过掺入 将荧光显微镜下观察到的荧光信号导入SCP-LVC-MS光学检测和细胞分离系统中。 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、高通量的技术空白， 分析单细胞中的代谢物、脂质和药物。这项研究的完成将使基础 生物医学的问题要追求，不能在此之前，如细胞内代谢物浓度如何 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。 像这样的基本问题对我们如何理解疾病，单细胞化学如何联系 跨越生物尺度（例如，器官水平表型）以及医学的许多其他基本方面。

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.