Context rich mass spectrometry of molecular localization and cellular interactions

摘要 细胞的分子组成和细胞与细胞的接触是如何随着时间或实验后发生变化的？ 操纵？这个基本问题贯穿于生物医学科学的各个领域， 大多数研究集中在确定生物现象的细胞和分子机制。然而， 我们回答这个问题的能力受到当前检测方法的局限性的严重限制。 特定细胞结构的蛋白质组成。想象一下生物科学的快速发展， 在每个实验中，我们可以在细胞的特定位置检测到多于一小部分的蛋白质。的 我们的项目的目标是开发一种新的方法来实现这一目标，称为"上下文丰富的质量 流式细胞术"-一种能够在细胞内进行蛋白质多参数分析的技术， 交互. 质谱法最近已成为细胞分析的有力工具。这项技术的一个版本 称为质量细胞术或CyToF的方法用于单细胞中蛋白质表达的多参数分析。是 类似于流式细胞术，因为细胞用抗体标记并在高浓度下逐个细胞地分析。 吞吐量的方式。然而，不像流式细胞术，其采用荧光标记的抗体， 由于限于~12个参数，质谱细胞术采用稀土金属作为抗体标记，并可用于 分析多达60种细胞内或细胞表面标记物。虽然是一项令人兴奋的技术，但大规模细胞计数需要 细胞从其天然微环境中提取并均质化成单细胞悬浮液， 分析.这使得将蛋白质签名与微环境背景联系起来非常具有挑战性。 我们的项目的目标是开发"上下文丰富的质谱仪"-一种技术，使多个- 在细胞相互作用的背景下蛋白质的参数分析。一旦发展出这种质谱分析法 方法将用于确定免疫突触内蛋白质的组成和组织。 这种由我们的技术所带来的新知识在未来可能会被用于策略和疗法 预防感染。

ABSTRACT How does the molecular composition of cells and cell-cell contacts change over time or after experimental manipulations? This fundamental question is asked across all fields of biomedical science and is at the heart of most studies focused on determining cellular and molecular mechanisms of biological phenomenon. Yet, our ability to answer this question is severely constrained by the limitations of current methods to detect the protein composition of particular cellular structures. Imagine the rapid progress biological science could make if we could detect more than a handful of proteins at specified locations on cells in each experiment. The objective of our project is to develop a new approach to achieve this goal—called “context-rich mass cytometry” –a technology that enables multi-parametric analysis of proteins in the context of cellular interactions. Mass spectrometry has recently emerged as a powerful tool for cell analysis. One version of this technology called mass cytometry or CyToF is used for multi-parametric analysis of protein expression in single cells. It is similar to flow cytometry in that cells are labeled with antibodies and analyzed on cell-by-cell basis in high- throughput manner. However, unlike flow cytometry which employs fluorescently-labeled antibodies and is limited to ~12 parameters, mass cytometry employs rare earth metals as antibody labels and may be used to analyze up to 60 intracellular or cell surface markers. While an exciting technology, mass cytometry requires that cells be extracted from their native microenvironment and homogenized into single cell suspension prior to analysis. This makes connecting the protein signature to the microenvironment context very challenging. The objective of our project is to develop “context-rich mass cytometry” –a technology that enables multi- parametric analysis of proteins in the context of cellular interactions. Once developed this mass spectrometry approach will be used to determine the composition and organization of proteins within immune synapses. This new knowledge enabled by our technology may, in the future, be parlayed into strategies and therapies for preventing infections.