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

 描述(申请人提供)：在生物学领域，特别是在人类疾病生物学领域，对单细胞水平的理解是根本性的缺乏。这一知识差距既包括单个细胞如何发挥作用，也包括单个细胞如何结合和协作构建多细胞创造，例如有多少癌细胞联合起来形成肿瘤。传统上，生物分析主要是通过大量(即数千或数百万)细胞被破坏并混合在一起形成一个完全人工的结构来进行的：细胞裂解物。细胞裂解物是从大量存在于组织样本中或在培养中生长的异质细胞中制备的。裂解产物的生物分析是经过人工样品制备的许多异质细胞的融合平均值，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，通过许多实验方法的进步，单细胞分析(SCA)已经成为可能。由于其无与伦比的检测灵敏度，质谱仪(MS)是最有希望用于单细胞分析的分析方法之一。单细胞质谱仪(SCMS)和质谱仪成像(MSI)是一种新兴的技术，可能会在基础科学和生物医学研究中给生物分析科学带来革命性的变化。我们研制了一种小型化的采样和电离装置--单探针，能够在环境条件下实时进行单细胞质谱分析。这种单探针的采样尖端小于真核细胞(&lt；10微米)，可以插入单个活细胞中采样细胞内化合物，以便立即进行MS分析。单探针SCMS技术已经在活的单个细胞内产生了对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。单探针还成功地用于MSI实验，从动物组织的切片中重建物种(如脂质和细胞代谢物)的空间分布。我们的长期研究目标是完全建立基于单探针的SCMS和MSI技术，以便在生物分析中得到广泛应用。这项应用的目标是开发单探针MS技术，作为一流的工具，用于细胞中的定量SCMS(QSCMS)和基于MSI的肿瘤研究。这项研究目标将通过完成以下三个具体目标来完成：1)建立培养细胞系单个细胞内化合物的定量单细胞质谱仪(QSCMS)；2)使用单探针MS分析体外肿瘤中的单个细胞；以及3)对体外和动物来源的肿瘤进行单探针MSI测量。单探针MS是一项创新技术，因为它为单细胞分析和组织成像提供了新的实验能力。这项拟议的研究具有重要意义，因为一旦开发出单探针MS实验应用程序(即qSCMS和MSI)，将允许对单细胞和肿瘤生物学的新理解，这是目前无法实现的。

 DESCRIPTION (provided by applicant): 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 Single- probe 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.