Due to the constraints regarding cell growth, modification of ribosomal genes in vivo is challenging. The reconstruction of E. coli ribosomes in vitro will be an important step to engineer ribosomes. We are trying to reconstruct the ribosome biogenesis process in E. coli lysates by expressing all of the ribosomal components so that the transcription, translation, and assembly of ribosomal building blocks proceed cooperatively. For the improvement and the validation of this strategy, analytical tools for ribosomal proteins (r-proteins) are necessary. From this point of view, we tried to develop a highly-specific method for the quantification of all of the ribosomal proteins (r-proteins) by using selected reaction monitoring (SRM). The SRM methods will enable highly specific quantification of r-proteins even if their concentrations are very low in samples.
In this study, we analyzed r-proteins derived from purified E. coli ribosome, E. coli lysate, and r-protein-overexpressed E. coli lysate using nanoLC–MS/MS (LCMS-8060) and developed optimized SRM methods for each r-protein.
【Results & Discussion】
Using the SRM methods, we successfully quantified 41 of 54 nascent r-proteins synthesized in E. coli lysate. To improve production efficiencies of r-proteins, we optimized translation enhancer region of the plasmids encoding r-proteins. In conclusion, we successfully developed a highly-specific and comprehensive analytical method for r-proteins, which will promote the study of ribosome reconstruction as well as the understanding of ribosome biogenesis.