Mass spectrometry-based methods for the comprehensive analysis of protein interactions in cells

蛋白质构成了任何类型细胞的大部分内容。不足为奇的是，它们也是主要的分子基团，负责执行细胞中的大多数任务，控制它们的形状和功能。为了确定蛋白质的功能，它们必须与其他分子进行物理上的相互作用，最常见的是其他蛋白质。这需要在细胞中建立一个复杂的蛋白质相互作用网络。有一些方法可以根据细胞对所带来的变化的反应来分析细胞，方法是识别所有相对数量发生变化的蛋白质，或者识别哪些其他蛋白质与选定的蛋白质相互作用。前者同时分析所有蛋白质，而后者只能研究在分析前已被定义为目标的蛋白质。目前，还没有一种方法能够分析所有蛋白质如何相互作用，或者这些相互作用如何随着细胞内外的变化而变化。我们之前开发了一种技术，可以研究细胞内的蛋白质相互作用。通过向活细胞中添加一种化学物质，它们就会“冻结”在各自的状态中，从而保持单个蛋白质的相互作用。然后，基于串联质谱学的生物分析技术可以用来确定单个蛋白质的相互作用以及参与其中的每个分子的身份。我们现在建议改进和优化这项技术，这样它就可以用于确定细胞中一整套蛋白质相互作用及其变化方式。这不仅将促进我们对细胞功能的了解，而且有朝一日还可能被用来研究导致植物、动物和人类疾病的内部和外部影响，并测试预防它们的对策。

Proteins make up the majority of the content of any type of cell. Not surprisingly, they are also the main group of molecules responsible for carrying out the majority of tasks in cells that govern their shape and functionality. In order for the proteins to ascertain their function, they have to physically interact with other molecules, most often other proteins. This requires a complex network of protein interactions to be established in cells. Methods are available that can analyze cells based on how they respond to changes brought upon them either by identifying all of the proteins that change in their relative amounts, or by identifying which other proteins interact with a selected protein. While the former method analyzes all proteins at the same time, the latter can only study the protein that has been defined as target prior to the analysis. Currently, no method exists that would be capable of analyzing how all proteins interact with each other, or how these interactions change in response to changes inside and outside of the cell. We have previously developed a technique that can study protein interactions inside cells. By adding a chemical to live cells, they are "frozen" in their respective state, which preserves individual protein interactions. Bio-analytical techniques that are based on tandem mass spectrometry can then be used to determine individual protein interactions and the identity of each molecule that participates in them. We are now proposing to refine and optimize this technique so it can be used for the determination of the complete set of protein interactions in cells and how they change. Not only would this advance our understanding of how cells function, it could also one day be used to study internal and external influences that cause diseases in plants, animals, and humans, and to test countermeasures to prevent them.