AbstractRibosomes 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 (r-proteins) by combining selected reaction monitoring (SRM) and 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 engineered ribosomes with additional functions.
Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins
