From Single Cells to Tissues: a Novel Mass Spectrometry Approach for Bioanalysis

在单细胞水平上，尤其是在人类， 疾病生物学这种知识上的差距既包括单个细胞如何发挥作用，也包括单个细胞如何发挥作用。 细胞联合收割机结合并协作构建多细胞创造物，例如有多少癌细胞与 形成肿瘤传统上，生物分析主要是通过大量（即数千或更多个）样品进行的。 数以百万计的细胞被破坏并混合在一起，形成一个完全人工的结构：细胞裂解物。 细胞裂解物由大量异质细胞制备，所述异质细胞存在于组织样品中，或存在于组织样品中。 在文化中成长。裂解物的生物分析是许多异质细胞的混合平均， 人工样品制备，因此，标准的生物分析方法在很大程度上无法 定义单细胞生物学。最近，单细胞分析（SCA）已经通过以下方面的进步而成为可能： 许多实验方法。由于其无与伦比的检测灵敏度，质谱法（MS）是一种 最有前途的分析方法将部署用于单细胞分析。单细胞质谱 （SCMS）和质谱成像（MSI）是新兴的技术， 基础科学和生物医学研究中的生物分析科学。我们开发了一种小型化的采样方法 和电离装置，单探针，能够进行单细胞质谱（SCMS）在真实的 在环境条件下的时间。单探针，取样尖端小于真核细胞（<10 μm）， 可以插入到单个活细胞中，对细胞内化合物进行取样，以立即进行MS分析。 单探针SCMS技术已经产生了细胞内化合物的高灵敏度检测， 包括细胞代谢物、脂质和抗癌化合物。单探针具有 也被成功地用于MSI实验，以重建物种（如脂质）的空间分布 和细胞代谢物）。我们的长期研究目标是全面建立单探针 基于SCMS和MSI的技术，可广泛用于生物分析。本申请的目的 是开发单探针MS技术，作为细胞定量SCMS（qSCMS）的一流工具 以及基于MSI的肿瘤研究。这一研究目标将通过完成 以下三个具体目标：1）建立化合物的定量单细胞质谱（qSCMS） 利用单探针质谱仪对体外培养的单细胞进行分析 肿瘤;和3）进行体外和动物来源的肿瘤的单探针MSI测量。的 单探针质谱是一项创新技术，它为单细胞质谱提供了新的实验能力， 分析和组织成像。由于单探针质谱实验 应用程序（即qSCMS和MSI），一旦开发，将允许新的理解单细胞和肿瘤 生物学是目前不可能的。

There is a fundamental lack of understanding in biology on the single cell level, especially in human disease biology. This gap in knowledge encompasses both how individual cells function and also how single cells combine and collaborate to construct multi-cellular creations, such as how many cancer cells associate to form a tumor. Traditionally, bioanalysis has largely been conducted through large numbers (i.e. thousands or millions) of cells being destroyed and mixed together to form a completely artificial construct: the cell lysate. Cell lysates are prepared from a large number of heterogeneous cells, either present in tissue samples or grown in culture. Bioanalysis of lysates are an amalgamated average of many heterogeneous cells subjected to artificial sample preparation, and therefore, standard bioanalytical methods have been largely unable to define single cell biology. Recently, single cell analysis (SCA) has become possible through advancements in many experimental methods. Due to its unparalleled detection sensitivity, mass spectrometry (MS) is one of the most promising analytical methods to be deployed for single cell analysis. Single-cell mass spectrometry (SCMS) and mass spectrometry imaging (MSI) are emerging techniques that could potentially revolutionize bioanalytical science in basic scientific and biomedical research. We have developed a miniaturized sampling and ionization device, the Single-probe, capable of performing single cell mass spectrometry (SCMS) in real time under ambient conditions. The Single-probe, with a sampling tip smaller than eukaryotic cells (<10 μm), can be inserted into individual living cells to sample the intracellular compounds for immediate MS analysis. The Single-probe SCMS technique has produced highly sensitive detection of intracellular compounds, including cell metabolites, lipids and anticancer compounds, inside living single cells. The Single-probe has also been used successfully in MSI experiments to reconstruct the spatial distribution of species (e.g. lipids and cell metabolites) from sections of animal tissue. Our long-term research goal is to fully establish the Singleprobe based techniques in SCMS and MSI for broad utilization in bioanalysis. The objective of this application is to develop the Single-probe MS technology as a first-in-class tool for quantitative SCMS (qSCMS) in cells and for MSI-based investigation of tumors. This research objective will be accomplished through completing the following three Specific Aims: 1) create quantitative single cell mass spectrometry (qSCMS) for compounds inside of single cells from cultured cell lines; 2) use the Single-probe MS to analyze single cells in in vitro tumors; and, 3) perform Single-probe MSI measurements of the in vitro and animal-derived tumors. The Single-probe MS is an innovative technology in that it provides new experimental capabilities for single cell analysis and tissue imaging. The proposed research is significant because the Single-probe MS experimental applications (i.e. qSCMS and MSI), once developed, will allow new understandings in single cell and tumor biology that are not currently possible.