Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) based on data-dependent acquisition (DDA) have been widely used to understand the biogenesis and function of ribosomes. However, DDA-based qMS sometimes does not provide the reproducible and quantitatively reliable analysis that is needed for high-throughput hypothesis testing. To overcome this problem, we developed a highly sensitive, specific, and accurate method to quantify all ribosomal-proteins by combining selected reaction monitoring (SRM) and stable isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates, and verified this approach as a high-throughput analytical tool by detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a high-throughput hypothesis testing tool in the research of E. coli ribosomes, and they have potential to accelerate the understanding of ribosome biogenesis, function, and the development of ribosome engineering.

中文翻译：

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选定的反应监测，用于定量大肠杆菌核糖体蛋白

核糖体是负责细胞中蛋白质合成的复杂机制。近年来，基于数据依赖采集（DDA）的定量质谱（qMS）已被广泛用于理解核糖体的生物发生和功能。但是，基于DDA的qMS有时无法提供高通量假设检验所需的可再现且定量可靠的分析。为了克服这个问题，我们开发了一种高度灵敏，特异且准确的方法，通过结合选择的反应监测（SRM）和稳定的同位素标记来定量所有核糖体蛋白。我们使用纯化的核糖体和大肠杆菌裂解物优化了SRM方法，并通过检测在大肠杆菌S30提取物中分别合成的54种r蛋白中的41种，验证了该方法作为高通量分析工具。